Wilhelm Roux' Archiv 161, 325--335 (1968)

Developmental Features and Synthetic Patterns of Male Germ Cells of Urechis caupo*

NIICMAL K. DAS

Department of Zoology, University of California, Berkeley

Received May 12, 1968

Summary. The process of spermategenesis in the echiuroid marine worm Urechis caupo takes place in the coelomic cavity. The nuclei of primary spermatocytes become gradually condensed from the G 1 to G 2 stages. The autoradiographic results show that the rate of RNA synthesis in these cells decreases considerably; no appreciable R~A synthesis is detected in cells in the Ge stage, meiosis, and in various stages of spermiogenesis. Synthesis of proteins, such as histones, protamines and also acrosomal basic protein, occurs during spermatogenesis when little or no RNA is made.

Zusammen]assung. Im marinen Wurm Urechis caupo findet die Spermatogenese im Coelom start. Die Kerne der prim~ren Spermatozyten werden yon der G l zur G 2 phase allm~hlich kondensiert. Unsere autoradiographischen Resultate zeigen, daI] die RNS Syn- theserate in diesen Zellen bedentend abnimmt; in Zellen der G 2 phase, der ]Y[eiose und der Spermiogenese wird keine nennenswerte I~NS Synthese beobachtet. Synthese yon Proteinen (Histone, Protamine und akrosomale basische Proteine) finder in der Spermatogenese statt, wenn wenig oder keine RNS synthetisiert wird.

In t roduct ion Al though i t has been observed in earl ier s tudies (see N~wBY, 1940) t h a t the

m a t u r a t i o n of male germ cells in Urechis caupo occurs in the coelomic cavi ty , such cells as spermatogonia and spe rmatocy tes have no t been def ini te ly ident i f ied; the detai ls and t imings of spermatogenic s tages are also no t known. This pape r deals wi th some descr ipt ive aspects of spermatogenesis in Urechis caupo; in addi t ion , the in ter re la t ionships among macromolecular syntheses dur ing spermabo- genesis in this organism have also been s tudied b y cy tochemica l methods , in an a t t e m p t to e lucidate fur ther t h e phys io logy of spcrmatogenesis . Similar s tudies on syn the t ic ac t iv i t ies during spermatogenesis in o ther animals and dur ing microsporogenesis in p lan t s have been repor ted b y several workers (TAY- Lor , 1959; HOTTA and STERN, 1963; H]~ND]~SO~, 1964; MVCKENTHALER, 1964; MON]~SI, 1965; DAS et al., 1965a). The resul ts p resented here will show t h a t in Urechis the process of spermatogenesis , beginning f rom one or more spermato- gonial mitoses to the end of spermiogenesis, t akes place in the coelomic cav i ty ; the male germ cells of th is organism synthesize pro te ins and go th rough meiosis and o ther s tages of m a t u r a t i o n when l i t t le or no new R N A is made.

Materials and Methods The male echiuroid marine worms, Urechis caupo, were kept in aerated sea water at

12--14~ Samples of coelomic fluid were collected by hypodermic syringe (26G, 3/8th needle) at various times. They were fixed in bulk in acetic alcohol or smeared on slides,

* This study was supported by a grant from the Iqational Science Foundation. I would like to thank Prof. M~x ALFERT for his advice and criticism and Mrs. JULIE

MIeou-E~sTwooD for help during these experiments and during the preparation of the manuscript.

326 N.K. DAs:

exposed to acetic alcohol or formMin vapor to preserve cell shapes, and post-fixed in acetic alcohol. These cells were then stained with Feulgen. In a few cases unfixed cells were Mlowed to swell in 0.2 M sodium chloride solution for 2 minutes or longer and then squashed in aceto- carmine to reveal meiotic stages. Frequencies of cell clusters in various stages of development were determined from smears of samples collected over a period of about two months. Cells with relatively large nuclei (Figs. 1 and 2) and nuclei in various stages of condensation (Figs. 3--6) have been scored as spermatogonia and primary spermatocytes, respectively (see results). Although it is likely that some spermatocytes with diffuse chromatin (Fig. 3) may have been scored as spermatogonial cells because of their size increase during the pre- paration of smears, the morphological and staining differences between spermatogonia and the rest of the spermatocytes were distinct. Nuclear Feulgen-DNA contents of these various cell types were measured by the two-wavelength cytophotometric method (PATAV, 1952) to determine the position of cells at different stages of interphase and also to distinguish between primary and secondary spermatoeytes.

In order to label DNA synthesizing cells 75 ~zc of tritiated thymidine (spec. act. 1.9 c/raM) were injected into the coelomic cavity of each worm. Samples of coelomic fluid (0.25 ml each) were then collected at different times from 1 day up to 49 days. To study RNA synthesis in male germ ceils, fresh coelomic fluid (0.5--1.0 ml) containing various types of cells was incubated in vitro at 20--24~ for 15 minutes in trit iated uridine (150 ~zc/ml; spec. act. 7.77 c/raM). (Coelomic fluid samples kept i~, vitro for more than a day at 20~ show no mor- phological distortion of various types of cells.) Samples in both experiments were fixed in the same manner described above. Samples of coelomie fluid were also exposed to various trit iated amino acids; the proce&tres for labeling, fixing, and staining of these cells have already been described in an earlier report (DAs and ALFnRT, 1968).

Autoradio~aphs were prepared by using liquid emulsion (NTB2). While cells exposed to H~-thymidine showed localization of label mainly in DNA, cells exposed to tta-uridine contained label in both I~NA and DNA, as was evident from nuclease digestion (see also DAs et aI., 1956b). Before autoradiographs were prepared, ceils labeIed with Ha-uridine and fixed in acetic alcohol were first stained with methyIgreen to identify various cell types (see DAS and ALlege, 1968, for methods of identification of primary spermatoeytes at dif- ferent stages of interphase). These cells were photographed and then digested with DNase after removing methylgreen in 45 % acetic acid and washing several times in distilled water. Cells IabeIed with trit iated thymidine or amino acids were stained with Feulgen prior to coating with emulsion (see also DAs and ALF~RT, 1968); cells labeled with Ha-uridine and digested with DNase were stained with Harris' hematoxylin through the processed emulsion.

Frequencies of various clusters of cells labeled with Ha-thymidine were determined. The rate of incorporation of Ha-uridine in nuclei or Ha-arginine in whole cells was studied by counting silver grains. The following procedures were used to determine whether the removal of nuclear protamine from maturing sperm (see D~s et al., 1967) by hot trichloro- acetic acid (TCA) results in a loss of radioactivity from these cells. Autoradiographs of samples, labeled for 30 minutes with H3-arginine and smeared on slides, were prepared and exposed for 18 horn's; clusters of heavily labeled spermatocytes and maturing sperm were mapped and grains were counted. Grains were then removed in solutions of potassium ferricyanide (0.1%) and Kodak acid fixer (F-5). Gelatin was dissolved in acetic acid (45%). These slides were washed for 10 minutes in hot (45--50~ tap water, hydrolyzed for 15 minutes in 5% TCA at 90~ washed thoroughly in water, and coated again with emulsion; the slides were exposed for 18 hours and grains were counted over the same mapped clusters.

Results

1. Spermatogenesis

Var ious t y p e s of f l e e - f l oa t i ng cell c lus ters a re p r e s e n t in t h e coe lomic f lu id of m a l e Urechis caupo a n d all cells in a n y one c lus te r a re s imi la r w i t h r e spec t

to t he i r n u c l e a r a n d cell size ( N ~ w n u 1940). P a r t s of such c lus te r of cells a re s h o w n in Figs . 1 - -8 . T h e nuc le i of t h e l a rges t cells a re d i f fuse ly s t a ined w i t h F e u l g e n (Fig. 1). Clus ters of sma l l e r cells a t i n t e r p h a s e w i t h r e l a t i v e l y d i f fuse

Male Germ Cells of Urechis caupo 327

chromatin (Fig. 3), condensing chromatin (Fig. 4), and with highly condensed ehromatin (Fig. 5) are also present. The presence of clusters of early spermatid (Fig. 8, ES), mature sperm (Fig. 8, MS), and sperm in various stages of matura- tion can be recognized from the degree of their nuclear condensation. Coelomie fluid also contains free-floating individual mature sperm, blood cells, and groups of somatic cells, called amoeboid cells (see N]~w~Y, 1940). Cells such as those shown in Figs. 1 and 3--5 were thought to be spermatids by N]~w]3Y (1940), since they gradually seemed to decrease in size, their nuclei became highly con- densed and no clusters of cells in division were observed. The cytophotometrie Feulgen-DI~A content of five nuclei from each cluster show that these cells are not spermatids; compared to the 1C (2.0d=0.1) DNA content of mature sperm nuclei, the DNA content of cells with diffuse (Fig. 3) and condensed (Fig. 5) chromatin are 2C (4.7 4-0.2) and 4C (9.74-0.6) amounts, respectively. Cells with condensing chromatin (Fig. 4) and cells with relatively large nuclei (Fig. l) have DNA values (6.1 4-0.4 and 6.34-0.2, respectively) intermediate between 2C and 4C amounts; these cells are in the process of premeiotie DNA synthesis. I t will be shown below that clusters of interphase cells as shown in Fig. 1 are spermatogonia and those shown in Figs. 3--5 are primary spermato- cytes.

A few clusters show large cells in synchronous division (Fig. 2); these are apparently spermatogonial cells in mitosis, since these divisions do not shortly lead to the formation of spermatids (see below). Small cells with highly condensed nuclei also divide (Figs. 6 and 7), but their chromosome morphology is indistinct. These cells, however, are likely to be in the first and second meiotic divisions, respectively; the Feulgen-DNA contents of five nuclei from each of these two clusters are 4C (8.84-0.6 and 2C (4.8• amounts, respectively. Meiotic divisions in coelomic fluid samples can be demonstrated clearly by treating cells in hypotonic salt solution and then squashing them in acetocarmine stain (Figs. 9 and 10).

Sperm formation in Urechis is periodic rather than continuous, at least in worms kept in the cold room. This is apparent in Fig. 1 l, which presents the fre- quencies of various types of cell clusters determined from coelomie fluid samples taken from a worm at different times up to 49 days. A eomp.arison of the accu- mulation and depletion with time of various cell types in Fig. 11 also suggests strongly that cells as shown in Fig. 1 and Figs. 3--5 are spermatogonia and primary spermatocytes, respectively. As the frequency of spermatocyies begins to decrease, the frequency of sperm clusters increases. After the complete deple- tion of spermatocytes the frequency of sperm clusters begins to drop and by the 25th day all sperm clusters disappear; (sperm clusters break up after sperm are fully mature and the mature sperm are then stored in the storage sacs; see Ns~wBY, 1940). New populations of sperm clusters begin to appear only after the formation of new spermatocytes. During the first 24--25 days of a cycle spermatogonial cells accumulate and then they begin to disappear, due to the formation of spermatocytes. Mitotic cells, as shown in Fig. 2, are present during the time spermatogonial cells accumulate; at the same time spermato- eytes and spermatids are absent. The frequencies of all these three types of cells determined from samples collected at 49 days are about the same as those seen

328 N.K. DAs:

Figs. 1--8. Parts of clusters of cells from coelomie fluid of male Urechis caupo. Cells were smeared, exposed to formalin vapor, post-fixed in acetic alcohol, and stained with Feulgen. • 1250. Spermatogonial cells in interphase (Fig. 1) and in mitosis (Fig. 2). Primary spermato- eytes in interphase (Figs. 3--5) and in first meiotic division (Fig. 6). Spermatocytes in second meiotic division (Fig. 7), early spermatids (Fig. 8, ES), and mature sperm (Fig. 8, MS)

a t the s t a r t of the exper iment . The t imes necessary for the accumula t ion of spermatogonia l cells, sperma~oeytes , and spe rm are 24 days (day 0 to 24 days) ,

Male Germ Cells of Urechis caupo 329

21 days (day 21 to day 42), and 24 days (day 0 to day 14 plus day 39 to day 49), respectively.

The identification of these cell types is further borne out by labeling cells with H3-thymidine at the DNA synthetic stage and then allowing them to go through the different stages of spermatogenesis. These results are presented in Figs. 12 and 13. Tritiated thymidine was used up during the first day after the injection in the coelomic cavity, since there was no increase in grain densities over labeled nuclei of cells fixed 2 days or later. I t can be seen in Fig. 12 that more than 60 % of spermatogonial cells and primary spermatocytes at interphase are labeled during the first day after Ita-thymidine injection. During the next

Figs. 9 and 10. Spermatoeytes in pachytene (Fig. 9) and in late prophase I, possibly diakinesis (Fig. 10). Unfixed cells were allowed ~o swell in 0.2 M NaC1 solution and squashed in aceto-

carmine. • 1530

two days all spermatocytes are labeled and they remain labeled until depleted at 16--18days (el. Fig. 11). The frequency of labeled spermatogonial cells decreases after about 1 day. The early spermatids start to become labeled in about 4 days and during the next 4 days all of them are labeled. The mature sperm clusters begin to show labeling 8 days after the injection of H3-thymidine and 6 days later all sperm are labeled. Among the dividing cells, shown in Fig. 13, only spermatogonial cells in mitosis (as in Fig. 2) show 100% labeling during the first day; this is expected because the duration of the G 2 (post-DNA syn- thetic interphaso stage) period of mitotic cells is usually shorter than that of the meiotic cells of the same organism (see data of MUCKENT~ALEtr 1964). Labeling of cells in the first meiotic division appears on the second day when most of the spermatoeytes in inter'phase are labeled (cf. Figs. 12 and 13). All early spermatids become labeled only after cells in the second meiotic division are labeled (cf. Figs. 12 and 13).

From these results with Ha-thymidine labeling, it is possible to estimate the duration of the period from meiosis to the end of spermiogenesis. This whole period takes about 10 days (time interval between 100% labeled spermatocytes to 100% labeled mature sperm in Fig. 12); about 6--7 days of this period are spent in the process of spcrmiogenesis (100 % labeled early spermatids to 100% labeled mature sperm). The remaining time (3--4 days) is required for meiotic divisions (100% labeled spermatocytes to 100% labeled early spermatids).

23a Wilhelm tLoux' Archly, Bd. 161

330 N.K. ])As:

I00- e - - � 9 : Spermotogoniol cells o - - o ; Spermotocytes x - - x : Sperm

so- ~

o 60- 2

3 "

20- i ~

\ o

0 8 16 24 32 40 48

T ime in doys

Fig. 11. Frequenc ies o f va r ious ce]| clusters in samples col lected a t d iEeren t t imes. The fixation and staining of cells are the same as s tated in the legend of Figs. 1 ~ 8 . Average

number of a l l clusters scored a t each point = 437

/ / ,/+ / / o tes

-E" I d / / x - - x :Early spermotids s ,~,--------" / 0 / z x - - - ~ :ao ture sperm , - - o :Mitotic

o - - o :lst Meiotic

40-

20-

04 - '~" '~-- , '9 ~ .... ' ' ' ' ~ . . . . . . ' ' -' ' ' 0 4 8 12 i6 20 ~ 0 2 4 6

Time (doys) of fer H S - T injection Time (doys) offer H 3 - T injection

Fig. 12 Fig. 13

Figs. 12 and 13. Frequencies of various labeled clusters of cells in interphase (Fig. 12) and in division (Fig. 13) as a function of t ime after the injection of ~S-thymidine (75 ~zc) in the coelomic cavity. The fixation and staining of cells are the same as s ta ted in the legends of Figs. 1--8. The average number of dusters of interphase and dividing cells scored a t each point are 100 and 20, respectively. Autoradiographs were exposed for 1--2 days. The data

in ]~igs. 11--13 were obtained from one worm

Male Germ Cells of Urechis caupo 331

2. R N A Synthesis

Cells exposed to Ha-uridine for 15 minutes show localization of label only in the nuclear RNA. The spermatogonial cells are as active as the somatic cells with respect to incorporation of tta-uridine (Table 1; Figs. 14a--15b). On the

Table i. Incorporation o/H3-uridine (150 ~c/ml) ]or 15 minutes in di]]erent cell types o/male Urechis. Various interphase stages o] primary 8permatocytes were estimated from nuclear conden- sation and staining (see DAs and ALFEI~% 1968). Autoradiographs were exposed ]or 3 days

Cell types No. of Average Grains cells grains in % of

per somatic nucleus cells

Somatic 45 53.9 100 Spermatogonial 45 53.6 99.5 Primary spermatocytes (G1) 45 18.9 35.1 Primary spermatoeytes (S) 45 3.5 6.5 Primary spermatocytes (G~) 45 0.2 0.4 First meiotic 57 0 - Second meiotic 30 0 - - Early spermatids 46 0 - - Immature sperm 51 0 - -

other hand, a gradual decrease in incorporation of HS-uridine into RNA takes place as nuclei of primary spermatocytes become condensed; compared to sper- matogonial or somatic cells, the rates of incorporation of HS-uridine in spermato- cytes at the G 1 (before DNA synthesis) and S (during DNA synthesis) stages are only 35% and 6% respectively (Table 1; Figs. 14a--17b). Spermatocytes at the G 2 stage show practically no incorporation of H3-uridine (Table 1 ; Figs. 16a and 16b). All meiotic cells, spermatids, and sperm do not incorporate tt3-uridine into RNA (Table 1).

3. Protein Synthesis

Protein synthesis in primary spermatocytes at various stages of interphase has been dealt with in our earlier studies, which showed that the incorporation of some tritiated amino acids in spermatocytes is about 2 4 times higher during the S-phase than during the G 1 or G 2 stage (DAS and ALF~T, 1968). These studies are now extended to other cell types. During a 30-minute labeling time, there is about 50% loss in incorporation of Ha-lysine in spermatocytes at the G 1 and G2 stages (grains/cell 2.8 • 0.4; N = 221) as compared to the incorporation in somatic cells (grains/cell 5.7 ~0 .7 ; N = 205). The incorporation of labeled amino acids continues to occur, although at a decreasing rate on a per cell basis, in meiotic cells and in maturing sperm (Table 2). After a 30-minute exposure to H3-arginine, some clusters of maturing sperm show heavier labeling than others. I t has been observed by using the alkaline fast green staining technique for histones (ALF~T and Gnsc~wIND, 1953) and also by analyzing amino acid con- tent (DAS et al., 1967) that a transition from somatic histones to protamine-like proteins occurs in maturing sperm nuclei of Urechis. The increased incorporation of Ha-arginine in some sperm groups may, therefore, be due to synthesis of

23*

332 N.K. DAS :

Figs. 14a--17b. Autoradiographs of different cell types labeled for 15 minutes withHa-uridine (150 ~xc/ml). Fig. a and b are photographs of the same cells before and after preparation of autoradiographs, respectively. Various interphase stages of primary spermatoeytes were estimated from nuclear morphology and staining intensity (see text ; also DAs and ALFE~T, 1968). Cells in Figs. a (14a--17a) were stained with methylgreen and cells in Figs. b (14b--17b) were stained with Harris' hematoxylin through the processed emulsion (see text). Auto- radiographs were exposed for 3 days. • 1300. Somatic cells (Fig. 14a and b), spermato-

gonial cells (Fig. 15a and b), and primary spermatocytes at G1 (Fig. 16a and b), G 2 (Fig. 16a and b), and S (Fig. 17a and b) stages

Male Germ Cells of Urechis caupo 333

Table 2. Incorporation o/ H3-arginine (50 ~c/ml) /or 30 minutes in spermatocytes and maturing sperm. Spermatocytes with condensing nuclei were considered to be at the S-phase (see DAs and ALFERT, 1968). Grains were counted over 2 ~ para//in sections o/d clusters o/meiotic cells and

10 clusters each o/ the other cell types. Autoradiographs were exposed/or 3 days

Cell types No. of No. of grains/ cells cell section

Primary spermatoeytes in S-phase 138 6.8=[=0.8 First meiotic 47 3.0=t=0.4 Maturing sperm 208 2.0•

Table 3. Loss o] radioactivity/tom spermatocytes and maturing sperm, labeled/or 30 minutes with Ha-arginine (50 ~c/ml), alter hydrolysis in trichloroacetic acid (TGA, 5 % for 15 min at 9O~ Only heaviest labeled clusters were used/or grain counts. Autoradiographs were exposed/or 18

hours (see text)

Cell types No. of Grains per cluster % grains clusters lost after

before after TCA TCA TCA

Spermatoeytes 11 59.2 4 4 . 6 26.9:1:6.6 Maturing sperm 11 45.8 1 3 . 0 73.8•

protamine-like protein. That most of the labeled proteins of these sperm nuclei are protamine-like is suggested by the fact that over 73 % of the radioactivity is removed following hydrolysis of these cells in hot TCA (Table 3). Protamines are known to be soluble in hot TCA (ALFEgT, 1956). Some loss of radioactivity from primary spermatocytes also occurs; this could be due to extraction of small fraction of lysine-rich histones (see H~n~IcA, 1967). Syntheses of protamines and arginine-rich histories have also been detected during spermiogenesis in other organisms (I~GLV~S etal . , 1966; BLOCH and BgACX, 1964; DAS etal . , 1964; MoNwsI, 1965).

Discussion

The present results show that similar to annelids and sipunculids (see M]~G- L~TSCH, 1967, p. 648), the process of spermatogenesis in the echiuroid worm Urechis caupo takes place in the coelomic cavity. Spermatogonial cells seem to be extruded into the coelomie cavity where they accumulate through mitotic divisions. These cells differentiate into primary spermatocytes. After a period of accumulation the primary spermatocytes enter meiosis, finally producing large number of sperm clusters from which individual sperm separate when fully mature and are then stored in the storage sacs (N~wBY, 1940). In contrast to oocytes, spermatocytes in meiosis were not seen by earlier workers (see N~wBY, 1940). This was mainly due to the unusually condensed state of the nucleus of these spermatocytes, since in the present study meiotic divisions are revealed by allowing nuclei to swell in hypotonic salt solution.

The primary spermatocytes of Urechis do not show an appreciable growth in size. There is a considerable decrease in RNA synthesis corresponding with the increase in ehromatin condensation. I t is known from earlier studies tha t

23b Wilhelm I~oux' Archly, Bd. 161

334 N.K. D~s :

condensed chromatin is relatively inactive in RNA synthesis (TAYLon, 1960; PRESCOTT and BENDEr, 1962; F~wNST~ et al., 1963; DAs et al., 1965a). Further- more, the nucleolus which is known to be an active site of RNA synthesis in the nucleus (see review by BvscH et al., 1963) is not seen in the condensing nuclei of Urechis spermatocy~es (DAs and ALFEn~, 1966). Cells in meiosis as well as in later stages of spermatogenesis appear inactive in RNA synthesis. These results are in contrast to those of earlier workers who found that spermatocytes of other organisms are highly active in RNA synthesis during all stages of premeiotic interphase; RNA synthesis also occurs, although at a decreasing rate, in the first and second meiotic prophase and early spermatids ( H ~ D ~ s o N , 1964; MUCKEN- ~ I ~ L ~ , 1964; ])AS etal., 1965a). I t has been shown by M o ~ s I (1965) that RNA synthesis ceases during the late interphase to early prophase I in the primary spermatoeytes of the mouse; synthesis resumes in these cells at the mid- pachytene. The role of RNA made during different stages of spermatogenesis is not known. During microsporogenesis in Trillium, ST~,~N and Honda (1963) observed that the progress of meiosis was delayed and various aberrant meiotic divisions were produced by blocking synthesis of RNA and proteins in particular. I t is clear from the present results tha t in Urechis, the initiation and completion of meiosis and the whole process of spermiogenesis occur in the virtual absence of RNA synthesis.

Synthesis of proteins continues to occur, although at a decreasing rate, in spermatogenie stages in which little or no RNA is made (see also DAs et al., 1965a). I t has been shown in our earlier studies that histones are made in primary spermatocytes of Urechis during the S-phase (DAs and AL~W~T, 1968). The present results show that this synthesis takes place at a time when the production of RNA is 93% below the normal rate. As in other organisms (IN~LwS et al., 1966), in Urechis nuclear protamine is made at the time RNA synthesis is completely absent. We have also observed that a basic protein accumulates in the aerosomes of maturing Urechis sperm (D~s et al., 1967). I t is likely that this acrosomal protein, like protamine, is also synthesized late in spermiogenesis when RNA synthesis is absent; exact data on the timing of this synthesis, however, are not available at the present time. These results, similar to those from other cell systems (see reviews by W~L~, 1967; NE~E~, 1967), indicate the existence o~ regulatory mechanisms which determine the time and place for the expression of stored genetic information during spermatogenesis in Urechis.

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Male Germ Cells of Urechis caul~o 335

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Dr. Nm•AL K. DAS Department of Zoology University of California Berkeley, California 94720, U.S.A.