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Cytological and cytofluorometrical studies on gametogenesis of Paragonimus ohirai (Trematoda:Troglotrematidae)
CYTOLOGICAL AND CYTOFLUOROMETRICAL STUDIES ON GAMETOGENESIS OF PARAGONIMUS OHIRAI (T~~TODA:TROGLOT~~TIDAE) YASUHIDE
ORIDO” and HIDEKAZLJ HATA?
* Department of Parasitology. School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-01, Japan t Department of Parasitology, Chiba University School of Medicine, Chiba 280, Japan
Abstract-ORrDo Y. and HATA H. 1988. Cytological and cytofluorometrical studies on gametogenesis of ~ur~go~jrn~~ o~jru~ (Trematoda:Troglotrematidae). lntertz~tjo~af ~o~rn~I for Pu~u~~f~~o~~1X: 95- 10 I. The male and female reproductive cells of ~~~~g~~~~~~ohirui were cytologically and cyto~uorometrically investigated throughout their development within both gonads. Spermatogonia proliferated by mitosis and were evaluated as diploid. Primary spermatocytes formed a “rosette” of eight cells, and the features of their chromatin illustrated the course of the first meiotic division. Their nuclei possessed doubled DNA content at every stage. Secondary spermatocytes at a sixteen-cell stage contained a diploid amount of DNA, while spermatids at a thirty-two cell stage and spermatozoa were haploid. The ovary contained female reproductive cells from oogonia to primary oocytes, with different sizes according to the level of development. The mature oocyte was characterized by a large nucleus with a prominent nucleolus and retieular chromatin, suggesting the nucleus to be at a diffuse stage. The oogonium was evaluated as diploid and the primary oocytes contained a double amount of DNA. INDEX KEY WORDS: Paragonimus ohirui; Digenea; trematode; cytology; cytofluorometry: genesis; oogenesis: meiosis; DNA content of nucleus: chromosome: chromatin.
INTRODUCTION P~~ug~~inz~s is hermaphroditic, as is the case with most trematodes, and the female gamete of P. ohirni is fertihzed by the sperm from its mating partner in the lung of the definitive host. By ultrastructural observations, Fujino, Ishii & Mori (1977) revealed the spermatogenesis of P. ohirai. Orido (1987) described the nuclear morphology of female reproductive cells during the course of development. Terasaki (1977) reported that the chromosomes numbered 22 univalents in mitosis and I 1 bivalents in GENUS
meiosis. However, the process of meiosis has not been sufficiently elucidated. Moreover, from the cytochemical aspect, the development of the reproductive cells of parasitic helminths has not yet been investigated. By the use of cytofluorometry, the DNA content of a nucleus can be readily determined. Of several methods, the improved 4’,6-diamidino-2phenilindole dihydrochIo~de (DAPI) staining method by Hamada & Fujita (1983) is both highly reliable and easy to perform. In the present studies, cytological and cytochemicai procedures were applied in order to clarify the process of gametogenesis of I-‘. ohirui. Especially, in the nuclei of the reproductive cells, the morphological
change and the transition described in detail.
spermato-
of DNA
content
are
MATERIALS AND METHODS Metacercariae of P. ohirui were obtained from the liver of the brackish water crab, Sesarmn dehaani. Adult worms were collected from worm cysts in the lung of a rat which had been infected with 25 metacercariae more than 10 weeks earlier. For the preparation of specimens, two methods were employed. One was a modified squashing method, as follows. Whole worms were fixed in Carnoy’s solution (methanol:acetic acid, 3: 1) overnight and preserved in 70% ethanol. After they were macerated in 45% acetic acid and swollen, the testes and ovary were dissected from the worm body. Each of these gonads was placed on a glass slide and torn into pieces to isolate their reproductive cells. The cells were spread by dropping the Carnoy’s solution on them and were then dried. As a supplement to the cytological examinations, fixed whole worms were stained with alcoholic hydrochloric acid-carmine, and then put through the above procedure (Snow, 1963). The other method for the specimens was the simple airdrying method of Terasaki & Nakamura (197X). The testes and ovary were removed intact from a live adult worm. Each of these gonads was put on a glass slide and torn into pieces in a 0.6% sodium citrate solution. After leaving it for 30 min. the slide was put into an enclosed chamber. The cells were first fixed by the evaporation of Carnoy’s solution in the
YASUHIDE ORIDO and HIDEKAZU Hara
96
so-called “rosette” of eight cells (Fig. 3). The nuclei of the spermatocytes were larger than those of the oogonia, and the features of their chromatin revealed that the cells were undergoing the first meiotic division (meiosis I). The stages of meiosis continued to advance with the synchronization of the rosette. In the nuclei at leptotene (Fig. 4) and zygotene. chromonemata were observed, with those of the former being thinner and longer than those of the latter. At the beginning of pachytene, they condensed to appear as thick chromosomal threads (Fig. 5). and 11 tightly paired bivalents of chromosomes wcrc shown at the latter stage of pachytene. The bivalentx split longitudinally through diplotene except for the portions of chiasmata, which were formed at one or several points on each chromosome. At diakincsis. this latter appearance had become rather prominent (Fig. 6). For the interpretation of this process, meiosis I, it was necessary for the specimens to be well spread out by the air-drying procedure. The secondary spermatocytes were sometimes observed as an aggregation of 16 cells with small nuclei at the completion of meiosis I (Fig. 7). The second meiotic division (meiosis II) resulted in a 32-cell stage of spermatids and thereafter, they differentiated to spermatozoa (Fig. 8). With the cytodifferentiation, the nuclei of the spermatids transformed into a comma-like shape in the sperm:)tozoa.
chamber for 30 min. Then Carnoy’s solution was dropped directly onto the cells, and they w’ere dried. For cytological examination, some of the specimens were stained with 10% Giemsa’s solution for 20 min. Apart from those procedures. the cells were observed in a living state by a Nomarski differential interference microscope for confirmation of their developmental process. The reproductive cells were well spread out by both specimen preparation methods, and were then prepared for DAPI staining according to the procedure of Hamada & Fujita (1983).The DAPI solution (SO ng/ml DAPI with Tris buffer. pH 7.4 containing 10 mM 2-mercaptoethylamine hydrochloride) was mixed just prior to the staining. The glass slide was immersed in the solution for 30 min at 4-C. and a cover glass was closely sealed with nail varnish to preserve the specimen in the DAPI solution. The fluorescence from a nucleus was observed when excited by ultraviolet light and was measured by a Nikon EFD epifluorescent microscope with a Pl microfluorometer. The measured value was compared with the mean value of the somatic cells which had undergone the same experimental procedures. From the specimens of the testes, the relative DNA content was examined in spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa. Moreover, the results of 50 spermatogonia at interphase, 50 primary spermatocytes in various developmental stages, and SO spermatozoa including spermatids were shown on a histogram. In the same manner, 150 female
reproductive cells were selected according to size (developmental stages). and similarly evaluated for their relative DNA content. RESULTS
Cytology of male reproductive cells The testes of P. ohirai contained
Cytology of female reproductive cells The female reproductive cells inside the ovary had large variations in size. The oogonium was small and possessed a round nucleus with dense chromatin (Fig. 9). The primary oocytes were observed in different stages of development, and their nuclei were extended proportionally (Fig. 1Oa-d). The chromatin at interphase was condensed and evenly distributed in the nucleus (Fig. 1 Oa). Then a thick thread of chromosomes was formed biased to one side of the nucleus
the male reproductive cells in various developmental stages from spermatogonia to spermatozoa. The spermatogonium was small and had a round nucleus, in which the chromatin was dense and diffuse (Fig. 1). It was independent or organized within a group of two or four cells by mitotic proliferation (Fig. 2a, b). The primary spermatocytes were found to be differentiated from the last stage of the oogonia and formed the
F~os. l-8. Photomicrographs showing spermatogenesis within the testes of Pumgoninnrs &hi. (Figs. I -2b, 4.7, X stained with DAPI and Figs. 5, 6 with Giemsa’s solution; Figs. I-2b, 4-6 prepared by the air-drying method and Figs. 7, X by the squashing method; Fig. 3 shown by a Nomarski differential interference microscope. Scale bar = 10 ,Um in Figs. l-5.7,X and 5 pm in Fig. 6.) FIG.
1, Spermatogonia
FIG. 2. (a) Four-cell
at a four-cell
stage spermatogonia
FIG. 3. Primary
spermatocytes
FIG. 4. Primary
of a primary
FIG. 7. Secondary
showing the formation
spermatocytes
FIG. 5. Pachytene FIG. 6. Diakinesis
spermatocyte
spermatids
of a “rosette”
showing leptotene
of primary
spermatocytes
FIG. 8. Developing
stage showing small and round diploid nuclei with diffuse chromatin.
showing at mitotic metaphase. (b) Mitotic chromosomes DNA showing twenty-two univalents.
spermatocytes
showingmeiotic
at a sixteen-cell and spermatozoa
amount
of eight cells. n, Nucleus; cp, cytophore.
nuclei with a double amount
showing thick threads chromosomes
with a double
of DNA.
of chromosomes.
with eleven bivalents
and chiasma
formation.
stage showing small nuclei with a diploid amount of DNA. (arrows)
showing comma-like
shaped haploid nuclei.
of
Gametogenesis
of Puragonimus ohirai
6
FIGS. 1-8.
98
YAWHIDE
ORIDO
(Fig. 1 Ob). It became more fibrous during the course of development (Fig. 1 Oc). The chromosomes of pachytene then organized a nucleolus from its own portion, and the rest were dispersed in the extending nucleus. The specimens revealed this process by staining with carmine and Giemsa (Fig. 11). The fully-developed oocyte had a large nucleus with a prominent nucleolus and rather reticular chromatin. With fluorescent staining, the nucleolus was exhibited as a relatively “cold” region, surrounded by strong fluorescence from the nucleolus-associated chromatin (Fig. 10d). Cytofluorometry of male and female reproductive cells Cytofluorometry was mainly used because, when the specimens were prepared by the air-drying method, it was found to be highly reliable from the point of view of its stability. However, it was sometimes difficult to measure the spermatocytes and oocytes in meiosis by this method because it scattered the chromatin and chromosomes. In such cases, a modified squashing method was utilized. Although
Ftos. 9-11, Photomicrographs squashing method and stained
and H~DEKAZUHATA
the relative DNA content was represented as arbitrary unit of fluorescence intensity (FU), reproductive cells were discriminated among haploid, diploid and tetraploid when the mean value of the measurement for somatic cells was settled at 20 FU and the estimated values of the reproductive cells were accumulated on the histogram. The histogram for the relative DNA content of the male reproductive cells exhibited three frequency peaks (Fig. 12). The first peak of about 10 FU was attained by the concentration of low FU values from the spermatids and spermatozoa. However, the values from the spermatogonia were variable. They were distributed over a wide ranged mid-portion of the histogram as well as part of the third peak. The values from the primary spermatocytes were about 40 FU and their accumulation formed the third peak. In the same manner, 150 female reproductive cells were measured on specimens prepared by the airdrying method, resulting in a histogram showing two peaks of frequency (Fig. 13). The measurement values from the oogonia were mostly settled around 20 FU
showing oogenesis within the ovary of k~~~,gonimu~ ohirai. (Figs. Y, 10 prepared by the with DAPI; Fig. 1I prepared by the air-drying method and stained with Giemsa’s solution. Scale bar = IO pm.)
FIG. 9. Oogonium
showing a small and round diploid nucleus with diffuse chromatin
FIG. 10. Primary oocytes in various developmental stages, such as (a) interphase, (b) early pachytene, (c) late pachytene and (d) the diffuse stage showing that the nuclei become larger during the course of development and the chromosomes at pachytene become fibrous toward the diffuse stage. The diffuse stage showing the nucleolus surrounded by strong fluorescence from the nucleolus-associated chromatin (arrow) and the dispersed other part of the chromatin. FIG. 1 1. Primary
oocyte at late pachytene
showing the nucleolus
formation
(arrow).
Gametogenesis
of ~urffg(~nimzz.~ ohirui
to form the first peak, while the remaining were scattered around 30 FU, and even toward 40 FU. On the other hand, the values from the primary oocytes were constant, irrespective of the stage of meiosis, and their accumulation formed a peak at about 40 FU. DISCUSSION
There have been numerous valuable reports on the gametogenesis of digenetic trematodes. Its process is characteristic among genus, even in species. However, meiosis, which composes part of the typical gametogenesis, has not been described in detail, especially in oogenesis. Various techniques for a cytological approach are required for the interpretation of meiosis in the male and female reproductive cells of P. ohirui. Hamada & Fujita (1983) carried out DAPI staining on the smears of somatic cells of a rat for cytofluorometry. Upon the present investigations of reproductive cells, the specimens prepared by the socalled squashing method and a kind of air-drying method were able to be used for cytofluorometry as well as for cytological studies. The stability of the measurement by the latter was thought to be due to the disappearance of the cytoplasm. The former method
had the advantage that fixed material could be utilized. In addition to cytological examination of the spermatogenesis of f. ohirni, Terasaki (1977) stated that the number of chromosomes was 22 in diploid and 11 in haploid. Fujino et nl. (1977) reported that a primary spermatogonium divided twice and became four tertiary spermatogonia. By cytofluorometry, the wide spread of the FU values from the spermatogonia at interphase was probably caused by the measurement for the nuciei which differ from the progress of DNA synthesis. Further division produced the eight-cell stage of the primary spermatocytes. In the prophase of meiosis I, the primary spermatocyte seemed to develop during the stages of leptotene, zygotene, pachytene, diplotene and diakinesis. The cyto~uorometric results showed that the nucleus of each stage possessed doubled DNA content. This suggests that most of the DNA for meiosis is synthesized in the interphase of meiosis I. The secondary spermatocytes were observed as the a~regation of sixteen cells at the completion of meiosis I. Although they were rarely found. several measurements by cytofluorometry were executed for
(N) 50 T
of the relative DNA content in the nuclei of the male reproductive cells of Purugnnimus ohirui, showing that the accumulation at about 10 FU, 20 FU and 40 FU occurred from the measurements for spermatozoa FIG. 12. The histogram including
spermatids,
spermatogonia at interphase and primary respectively. FU, Arbitrary unit of fluorescence
spermatocytes containing partial intensity; N, number of nuclei.
20 -. 10 !
.~.~ 0
10
20
30
40
il
spermatogonia.
!
50
mJ)
FIG. 13. The histogram of the relative DNA content in the nuclei of the female reproductive cells of Paragonimzzs ohirai, showing that the accumulation at about 20 FU occurred from oogonia at interphase and that at about 40 FU was from the primary oocytes containing partial spermatogonia. FU. Arbitrary unit of fluorescence intensity; N, number of nuclei.
100
YASLIHIOE
ORIDO
them. The results did show that the nuclei contained an amount of DNA equivalent to the diploid condition, although they carried a single set of chromosomes. Such a condition results from the separation of homologous pairs of duplicated chromosomes, occurring through the developmental course after metaphase, the latter stages of meiosis I. Meiosis II produced spermatids of a 32-cell stage from the secondary spermatocytes. Within the rosette of 32 cells, the spermatids differentiated to spermatozoa. The DNA content in their nuclei throughout spermiogenesis was constantly haploid. This evaluation in regard to spermatozoa corresponded to that of the mouse, which was measured by more complex methods of cytofluorometry (Pellicciari, Redi. Garagna, Fukuda & Manfredi Romanini, 1984; Redi, Garagna & Wottiroli, 1986). As mentioned above. the spermatogenesis of L? ohirrri was considered to be normal from both the morphological and cytochemical points of view. Compared with the numerous reports on spermatogenesis, oogenesis seems to have been investigated insufficiently. This may be because of the scarce cytomorphosis of the female reproductive cells during the course of their development within the ovary. It was reported that the nuclei of oogonia were smail and had condensed chromatin, while developing primary oocytes contained synaptonemal complexes in their nuclei, representing pachytene in meiosis I, and further developed oocytes possessed a large nucleus with a prominent nucleolus and dispersed chromatin (Orido, 1987). However, the correct identificati~~n of the stage classifications in meiosis have until now remained quite obscure. The nucleus of the oogonium with evenly dense chromatin was evaluated as diploid, although cytofluoromet~cai resuhs showed that the relative DNA content covered a wide range, leading to the same interpretation as for the results in spermatogonia. The primary oocyte at a very early stage was able to be discriminated from the oogonium by the size and morphology of the nucleus. The nucleus of the oocyte was enlarged throughout the course of meiosis I inside the ovary, and the features of the chromatin showed a unique developmental process (Figs. 1Oa-d and 1I): the evenly distributed chromatin at interphase formed a thick thread of chromosomes at pachytene and then became more fibrous toward the diffuse stage. Cytofluorometry supported the resuhs that the developing primary oocytes had constantly doubled DNA content in their nuclei. The nucleus of the fully-developed oocyte was characterized by a prominent nucleolus and reticular chromatin. In the oogenesis of Schisrosortrurilrnz donthitti, this type of oocytc is described as the “diffuse stage” (Nez & Short, 1957). Moreover, the diffuse stage preceded by pachytene is followed by diplotene in some kinds of plants (Mocns, 1964, 1968). Orido (1986) observed in the initial portion of the uterus of I’. ohirui that the primary oocyte formed
an<:I
HIDEKAZU
HATA
diplonema, with fertilization providing the turning point from the diffuse stage, and finally the unequal division of meiosis I produced a secondary oocyte. In addition to cestodes, some species of trematodes are also anomalous in gametogenesis. For example. L? westermmi has been reported to have two types of gametogenesis in which either normal bisexual reproduction or parthenogenesis of a triploid form occurs (Cho, Sasada & Takao, 1977: Terasaki, l980; Sakaguchi & Tada, 1980; Fujino & Ishii, 1982). P. ohirai was able to be cytologically identified with the former form of p, westermani. The specificities for the various types of gametogenesis are expected to be clarified by cytofluorometry. i2~lino~~led~~~rtre,Irs-Iam grateful to Professor F. Yasuzumi for offering the use of an epifluorescence microscope with a PI microfluorometer, and also to Dr. N. Okura for hit valuable advice. I sincerely thank Professor M. Oturu for his critical review of the manuscript.
REFERENCES
FUJI&O T.. ISHII Y. &
MORI ‘1‘. 1077. Ultrastructural stud& on the spermatozoa and spermatogeneais in rl’lrrrrgonin7~~ and Euqxrcma (Trematoda: Digenea). Japancx, Jo~~mrr~ ~fParu.~~~~~~b~26: 240-255. FUJIMI T. & 1~11 Y. 1982. Ultrastructur~i studie\ on spermatogenesis in a parthenogenetic type of I’crmgonimus ~vcmmuni (Kerbert, I X78) proposed as t’. phnonalis (Baelz. 1880). Jmrmal of Parasitology 68:
433-441. HAMADA
S. & FUJITA S. 1’983.
quantitative 226.
cyt~fluflr~metry.
MOENS I’. B. 1064. A
DAPI staining improved for ~j.~~oc~~~rjsl~ 79: 7 1il-
new interpretation of m&tic prophase in Lycqxmicon mc.rrlentun~ (tomato). C’hromosoma 15: 231-242. MOENS P. B. 196X. The structure and function of the svnaptinemal complex in Lilitcnz l~~~i~~)r~l~z sp0roqW.s. ~~~~~*~~.S~)F,~~~ 23: 4 18-45 1. NW M. M. 61 SI~ORT R. B. 1957. Ciametogcncsis in S~histcuomatilrm douthirti (Schistosomatidae: ‘frematoda). Journul of Parusitolog~~ 43: 167~ I X2. ORIDO Y. 19$h.‘l’he fine structure and the maturation of the octcvte of f’~l~~i~(~n~~?71~,~ ohimi. Japanr~ .f~)iir~i[l/ o/ f~~~~~~,s~i~~l~~~~ 35: suppl. 17 (in Japanese). OKIUO Y. 19X7. Development of the ovary and temale reproductive cells of the lung fluke, Parago~7in~rcsoh/rtri (~l‘rematod;~:Tro~lotrcmatid~~e). .Jofrrna/ o/ Parasitolqq\~ 73: lhl-171.
Gametogenesis
of Paragonimus ohirui
SAKAGUCHI Y. & TADA I. 1980. Karyotypic studies of lung flukes, Paragonimus iloktsuensis, P. sadoensis and I-‘. westermani, with special reference to gametogenesis in
P. westermani. Jupunese Journal of Parasitology 29: 25 I256. SNOW R. 1963. Alcoholic hydrochloric acid-carmine as a stain for chromosomes in squash preparation. Stain Technology 38: 9- 13. TERASAKI K. 1977.
Studies
on chromosomes
of the lung
flukes in Japan. Jupunese JournalofParasitology
101 26: 222-
229. TERASAKI K. 1980. Comparative observations on the development of germ cells between Paragonimus westermani (Kerbert, 1878) and P. pulmonalis (Baelz, 1880). Japanese Joumul of Parasitology 29: 127-I 36. TERASAKI K. & NAKAMURAT. 197X. A simple technique for chromosome studies in the flukes. Bulletin Azabu
Veterinuly College 3: 273-278.
ORIDO” and HIDEKAZLJ HATA?
* Department of Parasitology. School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-01, Japan t Department of Parasitology, Chiba University School of Medicine, Chiba 280, Japan
Abstract-ORrDo Y. and HATA H. 1988. Cytological and cytofluorometrical studies on gametogenesis of ~ur~go~jrn~~ o~jru~ (Trematoda:Troglotrematidae). lntertz~tjo~af ~o~rn~I for Pu~u~~f~~o~~1X: 95- 10 I. The male and female reproductive cells of ~~~~g~~~~~~ohirui were cytologically and cyto~uorometrically investigated throughout their development within both gonads. Spermatogonia proliferated by mitosis and were evaluated as diploid. Primary spermatocytes formed a “rosette” of eight cells, and the features of their chromatin illustrated the course of the first meiotic division. Their nuclei possessed doubled DNA content at every stage. Secondary spermatocytes at a sixteen-cell stage contained a diploid amount of DNA, while spermatids at a thirty-two cell stage and spermatozoa were haploid. The ovary contained female reproductive cells from oogonia to primary oocytes, with different sizes according to the level of development. The mature oocyte was characterized by a large nucleus with a prominent nucleolus and retieular chromatin, suggesting the nucleus to be at a diffuse stage. The oogonium was evaluated as diploid and the primary oocytes contained a double amount of DNA. INDEX KEY WORDS: Paragonimus ohirui; Digenea; trematode; cytology; cytofluorometry: genesis; oogenesis: meiosis; DNA content of nucleus: chromosome: chromatin.
INTRODUCTION P~~ug~~inz~s is hermaphroditic, as is the case with most trematodes, and the female gamete of P. ohirni is fertihzed by the sperm from its mating partner in the lung of the definitive host. By ultrastructural observations, Fujino, Ishii & Mori (1977) revealed the spermatogenesis of P. ohirai. Orido (1987) described the nuclear morphology of female reproductive cells during the course of development. Terasaki (1977) reported that the chromosomes numbered 22 univalents in mitosis and I 1 bivalents in GENUS
meiosis. However, the process of meiosis has not been sufficiently elucidated. Moreover, from the cytochemical aspect, the development of the reproductive cells of parasitic helminths has not yet been investigated. By the use of cytofluorometry, the DNA content of a nucleus can be readily determined. Of several methods, the improved 4’,6-diamidino-2phenilindole dihydrochIo~de (DAPI) staining method by Hamada & Fujita (1983) is both highly reliable and easy to perform. In the present studies, cytological and cytochemicai procedures were applied in order to clarify the process of gametogenesis of I-‘. ohirui. Especially, in the nuclei of the reproductive cells, the morphological
change and the transition described in detail.
spermato-
of DNA
content
are
MATERIALS AND METHODS Metacercariae of P. ohirui were obtained from the liver of the brackish water crab, Sesarmn dehaani. Adult worms were collected from worm cysts in the lung of a rat which had been infected with 25 metacercariae more than 10 weeks earlier. For the preparation of specimens, two methods were employed. One was a modified squashing method, as follows. Whole worms were fixed in Carnoy’s solution (methanol:acetic acid, 3: 1) overnight and preserved in 70% ethanol. After they were macerated in 45% acetic acid and swollen, the testes and ovary were dissected from the worm body. Each of these gonads was placed on a glass slide and torn into pieces to isolate their reproductive cells. The cells were spread by dropping the Carnoy’s solution on them and were then dried. As a supplement to the cytological examinations, fixed whole worms were stained with alcoholic hydrochloric acid-carmine, and then put through the above procedure (Snow, 1963). The other method for the specimens was the simple airdrying method of Terasaki & Nakamura (197X). The testes and ovary were removed intact from a live adult worm. Each of these gonads was put on a glass slide and torn into pieces in a 0.6% sodium citrate solution. After leaving it for 30 min. the slide was put into an enclosed chamber. The cells were first fixed by the evaporation of Carnoy’s solution in the
YASUHIDE ORIDO and HIDEKAZU Hara
96
so-called “rosette” of eight cells (Fig. 3). The nuclei of the spermatocytes were larger than those of the oogonia, and the features of their chromatin revealed that the cells were undergoing the first meiotic division (meiosis I). The stages of meiosis continued to advance with the synchronization of the rosette. In the nuclei at leptotene (Fig. 4) and zygotene. chromonemata were observed, with those of the former being thinner and longer than those of the latter. At the beginning of pachytene, they condensed to appear as thick chromosomal threads (Fig. 5). and 11 tightly paired bivalents of chromosomes wcrc shown at the latter stage of pachytene. The bivalentx split longitudinally through diplotene except for the portions of chiasmata, which were formed at one or several points on each chromosome. At diakincsis. this latter appearance had become rather prominent (Fig. 6). For the interpretation of this process, meiosis I, it was necessary for the specimens to be well spread out by the air-drying procedure. The secondary spermatocytes were sometimes observed as an aggregation of 16 cells with small nuclei at the completion of meiosis I (Fig. 7). The second meiotic division (meiosis II) resulted in a 32-cell stage of spermatids and thereafter, they differentiated to spermatozoa (Fig. 8). With the cytodifferentiation, the nuclei of the spermatids transformed into a comma-like shape in the sperm:)tozoa.
chamber for 30 min. Then Carnoy’s solution was dropped directly onto the cells, and they w’ere dried. For cytological examination, some of the specimens were stained with 10% Giemsa’s solution for 20 min. Apart from those procedures. the cells were observed in a living state by a Nomarski differential interference microscope for confirmation of their developmental process. The reproductive cells were well spread out by both specimen preparation methods, and were then prepared for DAPI staining according to the procedure of Hamada & Fujita (1983).The DAPI solution (SO ng/ml DAPI with Tris buffer. pH 7.4 containing 10 mM 2-mercaptoethylamine hydrochloride) was mixed just prior to the staining. The glass slide was immersed in the solution for 30 min at 4-C. and a cover glass was closely sealed with nail varnish to preserve the specimen in the DAPI solution. The fluorescence from a nucleus was observed when excited by ultraviolet light and was measured by a Nikon EFD epifluorescent microscope with a Pl microfluorometer. The measured value was compared with the mean value of the somatic cells which had undergone the same experimental procedures. From the specimens of the testes, the relative DNA content was examined in spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa. Moreover, the results of 50 spermatogonia at interphase, 50 primary spermatocytes in various developmental stages, and SO spermatozoa including spermatids were shown on a histogram. In the same manner, 150 female
reproductive cells were selected according to size (developmental stages). and similarly evaluated for their relative DNA content. RESULTS
Cytology of male reproductive cells The testes of P. ohirai contained
Cytology of female reproductive cells The female reproductive cells inside the ovary had large variations in size. The oogonium was small and possessed a round nucleus with dense chromatin (Fig. 9). The primary oocytes were observed in different stages of development, and their nuclei were extended proportionally (Fig. 1Oa-d). The chromatin at interphase was condensed and evenly distributed in the nucleus (Fig. 1 Oa). Then a thick thread of chromosomes was formed biased to one side of the nucleus
the male reproductive cells in various developmental stages from spermatogonia to spermatozoa. The spermatogonium was small and had a round nucleus, in which the chromatin was dense and diffuse (Fig. 1). It was independent or organized within a group of two or four cells by mitotic proliferation (Fig. 2a, b). The primary spermatocytes were found to be differentiated from the last stage of the oogonia and formed the
F~os. l-8. Photomicrographs showing spermatogenesis within the testes of Pumgoninnrs &hi. (Figs. I -2b, 4.7, X stained with DAPI and Figs. 5, 6 with Giemsa’s solution; Figs. I-2b, 4-6 prepared by the air-drying method and Figs. 7, X by the squashing method; Fig. 3 shown by a Nomarski differential interference microscope. Scale bar = 10 ,Um in Figs. l-5.7,X and 5 pm in Fig. 6.) FIG.
1, Spermatogonia
FIG. 2. (a) Four-cell
at a four-cell
stage spermatogonia
FIG. 3. Primary
spermatocytes
FIG. 4. Primary
of a primary
FIG. 7. Secondary
showing the formation
spermatocytes
FIG. 5. Pachytene FIG. 6. Diakinesis
spermatocyte
spermatids
of a “rosette”
showing leptotene
of primary
spermatocytes
FIG. 8. Developing
stage showing small and round diploid nuclei with diffuse chromatin.
showing at mitotic metaphase. (b) Mitotic chromosomes DNA showing twenty-two univalents.
spermatocytes
showingmeiotic
at a sixteen-cell and spermatozoa
amount
of eight cells. n, Nucleus; cp, cytophore.
nuclei with a double amount
showing thick threads chromosomes
with a double
of DNA.
of chromosomes.
with eleven bivalents
and chiasma
formation.
stage showing small nuclei with a diploid amount of DNA. (arrows)
showing comma-like
shaped haploid nuclei.
of
Gametogenesis
of Puragonimus ohirai
6
FIGS. 1-8.
98
YAWHIDE
ORIDO
(Fig. 1 Ob). It became more fibrous during the course of development (Fig. 1 Oc). The chromosomes of pachytene then organized a nucleolus from its own portion, and the rest were dispersed in the extending nucleus. The specimens revealed this process by staining with carmine and Giemsa (Fig. 11). The fully-developed oocyte had a large nucleus with a prominent nucleolus and rather reticular chromatin. With fluorescent staining, the nucleolus was exhibited as a relatively “cold” region, surrounded by strong fluorescence from the nucleolus-associated chromatin (Fig. 10d). Cytofluorometry of male and female reproductive cells Cytofluorometry was mainly used because, when the specimens were prepared by the air-drying method, it was found to be highly reliable from the point of view of its stability. However, it was sometimes difficult to measure the spermatocytes and oocytes in meiosis by this method because it scattered the chromatin and chromosomes. In such cases, a modified squashing method was utilized. Although
Ftos. 9-11, Photomicrographs squashing method and stained
and H~DEKAZUHATA
the relative DNA content was represented as arbitrary unit of fluorescence intensity (FU), reproductive cells were discriminated among haploid, diploid and tetraploid when the mean value of the measurement for somatic cells was settled at 20 FU and the estimated values of the reproductive cells were accumulated on the histogram. The histogram for the relative DNA content of the male reproductive cells exhibited three frequency peaks (Fig. 12). The first peak of about 10 FU was attained by the concentration of low FU values from the spermatids and spermatozoa. However, the values from the spermatogonia were variable. They were distributed over a wide ranged mid-portion of the histogram as well as part of the third peak. The values from the primary spermatocytes were about 40 FU and their accumulation formed the third peak. In the same manner, 150 female reproductive cells were measured on specimens prepared by the airdrying method, resulting in a histogram showing two peaks of frequency (Fig. 13). The measurement values from the oogonia were mostly settled around 20 FU
showing oogenesis within the ovary of k~~~,gonimu~ ohirai. (Figs. Y, 10 prepared by the with DAPI; Fig. 1I prepared by the air-drying method and stained with Giemsa’s solution. Scale bar = IO pm.)
FIG. 9. Oogonium
showing a small and round diploid nucleus with diffuse chromatin
FIG. 10. Primary oocytes in various developmental stages, such as (a) interphase, (b) early pachytene, (c) late pachytene and (d) the diffuse stage showing that the nuclei become larger during the course of development and the chromosomes at pachytene become fibrous toward the diffuse stage. The diffuse stage showing the nucleolus surrounded by strong fluorescence from the nucleolus-associated chromatin (arrow) and the dispersed other part of the chromatin. FIG. 1 1. Primary
oocyte at late pachytene
showing the nucleolus
formation
(arrow).
Gametogenesis
of ~urffg(~nimzz.~ ohirui
to form the first peak, while the remaining were scattered around 30 FU, and even toward 40 FU. On the other hand, the values from the primary oocytes were constant, irrespective of the stage of meiosis, and their accumulation formed a peak at about 40 FU. DISCUSSION
There have been numerous valuable reports on the gametogenesis of digenetic trematodes. Its process is characteristic among genus, even in species. However, meiosis, which composes part of the typical gametogenesis, has not been described in detail, especially in oogenesis. Various techniques for a cytological approach are required for the interpretation of meiosis in the male and female reproductive cells of P. ohirui. Hamada & Fujita (1983) carried out DAPI staining on the smears of somatic cells of a rat for cytofluorometry. Upon the present investigations of reproductive cells, the specimens prepared by the socalled squashing method and a kind of air-drying method were able to be used for cytofluorometry as well as for cytological studies. The stability of the measurement by the latter was thought to be due to the disappearance of the cytoplasm. The former method
had the advantage that fixed material could be utilized. In addition to cytological examination of the spermatogenesis of f. ohirni, Terasaki (1977) stated that the number of chromosomes was 22 in diploid and 11 in haploid. Fujino et nl. (1977) reported that a primary spermatogonium divided twice and became four tertiary spermatogonia. By cytofluorometry, the wide spread of the FU values from the spermatogonia at interphase was probably caused by the measurement for the nuciei which differ from the progress of DNA synthesis. Further division produced the eight-cell stage of the primary spermatocytes. In the prophase of meiosis I, the primary spermatocyte seemed to develop during the stages of leptotene, zygotene, pachytene, diplotene and diakinesis. The cyto~uorometric results showed that the nucleus of each stage possessed doubled DNA content. This suggests that most of the DNA for meiosis is synthesized in the interphase of meiosis I. The secondary spermatocytes were observed as the a~regation of sixteen cells at the completion of meiosis I. Although they were rarely found. several measurements by cytofluorometry were executed for
(N) 50 T
of the relative DNA content in the nuclei of the male reproductive cells of Purugnnimus ohirui, showing that the accumulation at about 10 FU, 20 FU and 40 FU occurred from the measurements for spermatozoa FIG. 12. The histogram including
spermatids,
spermatogonia at interphase and primary respectively. FU, Arbitrary unit of fluorescence
spermatocytes containing partial intensity; N, number of nuclei.
20 -. 10 !
.~.~ 0
10
20
30
40
il
spermatogonia.
!
50
mJ)
FIG. 13. The histogram of the relative DNA content in the nuclei of the female reproductive cells of Paragonimzzs ohirai, showing that the accumulation at about 20 FU occurred from oogonia at interphase and that at about 40 FU was from the primary oocytes containing partial spermatogonia. FU. Arbitrary unit of fluorescence intensity; N, number of nuclei.
100
YASLIHIOE
ORIDO
them. The results did show that the nuclei contained an amount of DNA equivalent to the diploid condition, although they carried a single set of chromosomes. Such a condition results from the separation of homologous pairs of duplicated chromosomes, occurring through the developmental course after metaphase, the latter stages of meiosis I. Meiosis II produced spermatids of a 32-cell stage from the secondary spermatocytes. Within the rosette of 32 cells, the spermatids differentiated to spermatozoa. The DNA content in their nuclei throughout spermiogenesis was constantly haploid. This evaluation in regard to spermatozoa corresponded to that of the mouse, which was measured by more complex methods of cytofluorometry (Pellicciari, Redi. Garagna, Fukuda & Manfredi Romanini, 1984; Redi, Garagna & Wottiroli, 1986). As mentioned above. the spermatogenesis of L? ohirrri was considered to be normal from both the morphological and cytochemical points of view. Compared with the numerous reports on spermatogenesis, oogenesis seems to have been investigated insufficiently. This may be because of the scarce cytomorphosis of the female reproductive cells during the course of their development within the ovary. It was reported that the nuclei of oogonia were smail and had condensed chromatin, while developing primary oocytes contained synaptonemal complexes in their nuclei, representing pachytene in meiosis I, and further developed oocytes possessed a large nucleus with a prominent nucleolus and dispersed chromatin (Orido, 1987). However, the correct identificati~~n of the stage classifications in meiosis have until now remained quite obscure. The nucleus of the oogonium with evenly dense chromatin was evaluated as diploid, although cytofluoromet~cai resuhs showed that the relative DNA content covered a wide range, leading to the same interpretation as for the results in spermatogonia. The primary oocyte at a very early stage was able to be discriminated from the oogonium by the size and morphology of the nucleus. The nucleus of the oocyte was enlarged throughout the course of meiosis I inside the ovary, and the features of the chromatin showed a unique developmental process (Figs. 1Oa-d and 1I): the evenly distributed chromatin at interphase formed a thick thread of chromosomes at pachytene and then became more fibrous toward the diffuse stage. Cytofluorometry supported the resuhs that the developing primary oocytes had constantly doubled DNA content in their nuclei. The nucleus of the fully-developed oocyte was characterized by a prominent nucleolus and reticular chromatin. In the oogenesis of Schisrosortrurilrnz donthitti, this type of oocytc is described as the “diffuse stage” (Nez & Short, 1957). Moreover, the diffuse stage preceded by pachytene is followed by diplotene in some kinds of plants (Mocns, 1964, 1968). Orido (1986) observed in the initial portion of the uterus of I’. ohirui that the primary oocyte formed
an<:I
HIDEKAZU
HATA
diplonema, with fertilization providing the turning point from the diffuse stage, and finally the unequal division of meiosis I produced a secondary oocyte. In addition to cestodes, some species of trematodes are also anomalous in gametogenesis. For example. L? westermmi has been reported to have two types of gametogenesis in which either normal bisexual reproduction or parthenogenesis of a triploid form occurs (Cho, Sasada & Takao, 1977: Terasaki, l980; Sakaguchi & Tada, 1980; Fujino & Ishii, 1982). P. ohirai was able to be cytologically identified with the former form of p, westermani. The specificities for the various types of gametogenesis are expected to be clarified by cytofluorometry. i2~lino~~led~~~rtre,Irs-Iam grateful to Professor F. Yasuzumi for offering the use of an epifluorescence microscope with a PI microfluorometer, and also to Dr. N. Okura for hit valuable advice. I sincerely thank Professor M. Oturu for his critical review of the manuscript.
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S. & FUJITA S. 1’983.
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of Paragonimus ohirui
SAKAGUCHI Y. & TADA I. 1980. Karyotypic studies of lung flukes, Paragonimus iloktsuensis, P. sadoensis and I-‘. westermani, with special reference to gametogenesis in
P. westermani. Jupunese Journal of Parasitology 29: 25 I256. SNOW R. 1963. Alcoholic hydrochloric acid-carmine as a stain for chromosomes in squash preparation. Stain Technology 38: 9- 13. TERASAKI K. 1977.
Studies
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101 26: 222-
229. TERASAKI K. 1980. Comparative observations on the development of germ cells between Paragonimus westermani (Kerbert, 1878) and P. pulmonalis (Baelz, 1880). Japanese Joumul of Parasitology 29: 127-I 36. TERASAKI K. & NAKAMURAT. 197X. A simple technique for chromosome studies in the flukes. Bulletin Azabu
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