ABSTRACT AN ULTRASTRUCTURAL STUDY OF SPERMATOGENESIS IN TWO SPECIES OF RANA BY Gary Raymond Poirier The morphology of spermatogenesis in two species of Rana (3. pipiens and E. clamitans) has been investigated with the electron microscOpe. Three distinct types of spermatogonia can be distinguished, one of which reaches sizes of 20 to 25 um in diameter. It is in these large cells that mitochondrial multiplication occurs. Struc- turally there appears to be no difference between the spermatogonia of the two species. Certain of the spermatogonia described here have characteristics similar to the type A and B spermatogonia of mammals. Meiosis, similar in both species studied, is described through prOphase of the first division. No secondary spermatocytes could be positively identified. The early phases of spermiogenesis are similar in both species. However, each species has a different medhanism for acrosome formation. In 3. clamitans, the ‘ ‘Ar‘ - ‘ - ="‘EZ..'3 .5 -V‘ g ' ‘ .V’.- .n'." .- 0. a: . -- .. u:..‘ ‘ an “V“ l :;:-::s' a..— Q ‘ 9....-. 'e ‘-‘ .II a: . —-.:. hut--. . --‘- .‘ e w .- Ivo'. o‘cou -v..-- a . . bar-z. - A“ "0nd vovo. O :‘Foa‘..'¢ U-.¢v-~o-‘ .'..'.’_, -5 F" - .ooovoboC-C -00- c ~.-. .‘VOJV-l: ' cl. ‘.. - " M 1... ..cr Fay- rv- ~ 2.- ‘v V. ‘ “U‘ “ h‘ e3-.-65 a: . ‘1 Q one Z;:~:s . - I. ~' ‘ s.b .' l . Flt-9 013'. I S. I. ‘. .:.ae' ha a " r9a5 .' _ o l';~ "'E “ s . OR “(a s "Hm”‘ré a H“ ‘31:“. 'h‘ I. D va':.‘5 to he 1.. ‘2 “v fiesta-u. . 5.". 1n LC... U A I ”I. a. I,‘ I. ‘\ ‘4‘ Gary Raymond Poirier acrosome is formed by the fusion of vesicles thought to be derived from a Golgi-like cisternal apparatus. In 1:. pipiens, acrosomal formation occurs at the time when Innclei are fully elongated and condensed; no Golgi or Golgirlike elements appear to be associated with its formation. Structurally the two acrosomes are completely different. The acrosomes of E. clamitans spermatozoa consist of homogeneous bag-like structures overlapping the anterior portion of the nuclei. The acrosomes of 5. pipiens spermatozoa are situated at the most anterior portion of the nuclei, with no overlap at the anterior portion of the latter. The acrosomes of this species consist of a large PAS-positive granule, membrane stacks and/or vesicles of various sizes and densities. The mid-pieces of both species are of the "primi- tive" type. The mitochondria are not fused but remain as individual organelles. The mitochondria in the mid-pieces of g. clamitans spermatozoa have many well developed cristae, whereas in g. pipiens the cristae in the mid- piece mitochondria are poorly developed and scarce. This is thought to be a true species variation. Two centrioles are present in both species, with the distal one being continuous with the axial filament. The tail consists of the simple 9 + 2 axial filament arrangement throughout its 9'": lei-05‘ a fi'..' I]: '0‘. :::.;:;'.'e tape cf C I ‘ an a R". Q ' D '13 1:5:. ‘9: ‘dl 6| I :'=*r=‘s of r' " :""" ’51: ' v 9 ';'~v- '1“. I ‘ - a fig F «inn/Cu ‘t‘ fitte S- I R' ‘ ‘ Orle 39"'e‘fir h— b .'O'- A . " ‘ 4...: 35'.” ‘ h ‘ ‘ b.-U.‘ ‘6. “"Q “o. ‘-. RA ‘ 6.... “A“ H . .U.l..|v‘. u: .....‘. U... . ‘ .~ ““5 :n" A”... b ' ‘Vu. .' . r, . J n‘ “or“: V“ s:""‘- ."3‘24 .‘ . vvfiohu .n ‘. A b“ V' V‘ ;-E. =.A A 0 N'Vzva ; Q ‘ I f : .-¢;‘ ‘n Gary Raymond Poirier entire length. R. clamitans spermatozoa also contain a primitive type of connecting piece. No similar structure was observed in R. pipiens spermatozoa. Various types of intranuclear inclusions, includ- ing packets of glycogen and mitochondria-like bodies, are described in the spermatids and spermatozoa of both species. The development of the follicle cell into the mature Sertoli cell is similar enough in both species to warrant a common description. Microtubules are associated with this development. Non-specific acid and alkaline phosphatases are located in the acrosomal membranes of R. clamitans spermatozoa. Alkaline phosphatase is also found in the add—pieces and axial filaments of R. clamitans spermatozoa. Localizations of alkaline and acid phosphatases were not carried out for R, pipiens spermatozoa. .~ ‘ 55 V. w? ‘ IO Q. AN ULTRASTRUCTURAL STUDY OF SPERMATOGENESIS IN TWO SPECIES OF RANA BY Gary Raymond Poirier A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1970 Iwi h to a A... .0 F..- .3: F 0-. ,4- -‘u 0.5, ray 6" 3. .-~': . ..‘ ..' .L..s s-‘-“ 'I no. ' ‘I Av I.. .P .2. n "" U.- ' V's no. I .3; 0“ my g‘ a ._ .; 1 25' :i'OIa has Lib: "s A 9“ 15" >". ‘uvla W- Va! .. ‘.'_ ‘ L ‘L 5,. R. ‘ ‘ , ~.' ‘ .- \§I .‘Q‘F’h A. re. 4" 0“" 4“. \‘C‘: 51.9 C01‘rse ACKNOWLEDGMENTS I wish to thank Dr. John R. Shaver for his suggestions, patience and understanding during the course of this study. His confidence in me and willingness to "go to bat” for me has made my graduate studies both fruitful and enjoyable. I am also grateful to Dr. Gordon Spink for his unselfish attitude in making his experience and laboratory facilities available for this study. My thanks to Drs. N. Band and H. Ozaki for their suggestions on various portions of this work and for serving on my graduate committee; to Mrs. June Mack and Mrs. Carola Wilson for juSt being Mrs. June Mack and Mrs. Carola Wilson; to Robert Click for being available to discuss problems as they arose and to Mrs. S. J. Rose for the line drawings. A special word of thanks goes to "Mac" who and whose help in a multitude of ways will always be remembered. I would also like to thank my parents, Mr. and Mrs. Ralph A. Poirer for their encouragement and aid during the course of my schooling. ii Lastly, I v " " A .. a“ a' .' ubuuobdd ‘0' I: n a - :O.:‘I.‘.;A' Av-yi‘fi O I 4 ~n..u.¢au Hub an? n. Lastly, I would like to express to my wife, Judy, my gratitude and appreciation for her self-sacrifice and dedication during the years in graduate school. iii p. n “I uvno‘D. " ' . O o . ‘“ bow-.— nut h..‘."‘-"\\‘ . u..,,.".. v. P. ‘QR vi :1 _.v ¥ n‘ T.“ u d . w IUCOIl. ..V.‘ V. A.‘ 1' mt: FO-QC‘O “v“ v. k , . . . n V‘ "“~-~..~ rh‘“ ""vau :.--‘.o‘ -. A“. 'uu d A ‘ u...g.|...- a. .— '_. ..A~.‘A~c'e= ~ - ‘ V“-.. ~:'.~A‘VV-J. ‘ In. 1 ‘._"‘ a... . v ‘ 0" & P‘.w‘ 0. . ‘ V". 9.“ ~ .V-t‘e 2 p.-. .‘." ‘. :;~A":.M Q'.GP~ Uni-Hue“. 'rv‘- Val-z'fla. .~.v=.s . . . ‘ . I .' " .ARA‘ -:v~--s.vfl:tges-s 4 ::P.', c“aha F‘V; ~n..' ”~b“"-vq. d ‘ d a n \I a. h u!€v.€a . . v- -::§H’es ”.7-3’2 ”w“ km... a b \‘CV. A" ’6: '!~-N‘Vu-.o»u a r::“": ‘nqv~ thaw-$.53 ..a‘: A ‘I ¢ \I .ANA.‘ a “(EH‘d‘Vfltu‘Ee v‘.._. F - '\ .~. e cue . A‘ " R; .‘»Q:" M‘ -r. ‘ - C .- ""V=V"3es C‘ ‘- ‘fl .- I F C - l. §U=V¢~ues C‘ : I ‘.“-\ ‘. ‘ ‘ ~ I. V tof‘fi 'e PE‘ .5 u‘l IA Q Q "I r‘ 'l \ Usssb§e ~5‘&E - \cv‘fi‘ ' r‘ s “W4 reds I ‘3-.. . §~ ‘ 5. ‘ ‘ s ‘1' 5" d. ....e: 5;: iry, Once a |.. ‘ V ‘e, l .P ...u in 3U.‘ ‘B u‘un Cf '0 “n n 1‘” vy“:" ! c.‘:c.s were ‘ U I. A "y =NA‘ n by... . “l?”- ‘ngH s .. u “33%. cli:s‘ ML $.Ae ‘. I.‘I“ ‘tdfi s 5 '3‘ P Wage | k v \‘. ~ ._‘ l6 375C prior to use. The controls consisted of incubating a portion of the samples in the above medium without sodium B—glycerolphosphate. Post-fixation with osmium tetroxide was carried out as described above. Light Microscopy Sections approximately l mu thick were taken from the same paraformaldehyde-gluteraldehyde-osmium fixed samples as those used for the electron microscopic inves- tigation. The thick sections were taken dry from the glass knives with fine tapered forceps and placed on a drop of distilled water on a microscope slide and allowed to air dry. Once dried the sections were stained with a 0.05% solution of toluidine blue in distilled water. All EmotOgraphs were taken on 35 mm film in a Zeiss or Wild photoscope . Periodic Acid-Schiff (PAS) The PAS procedure used was that described by Feder and O'Brien (1968). Aldehyde blockade was performed to Cover Schiff-positive groups introduced by fixatiOn. For this the slides were placed in a saturated solution of DNFH (2,4-dinitropheny1hydrazine) in 15% acetic acid for 10 minutes. The slideSJwere washed for 10 minutes in running tap water, then were placed in Schiff's reagent flHTZO minutes. Schiff's reagent was made according to . ‘ an IV“ ~flv d Q II: '.V' H“. eS‘ o a ‘ll' . 21.1.1: 5 1:85-1:11 Ill :0: :‘Iynnb‘V' in? J an doovvh. 60. I ‘.II I. III}! 50:1“: :6 Q l l 'l::fl Or 1"“. “R Qo-Uv‘ . A man..." ~I; lie-wt b ' - F... vv‘oouers balnt 17 the procedure described by Davenport (1960). After the Schiff's treatment the slides were transferred quickly and directly into three successive baths, 2 minutes each, of 0.05% sodium metabisulfate. Finally the slides were rinsed in running water for 5 minutes. Some sections were counterstained with toluidine blue as described above. OBSERVATIONS Spermatocytogenesis Two types of spermatogonia have been described at the. light microscopic level in a large number of amphibians by Champy (1913) . Three types were distinguished by Sharma and Sekhri (1955) in _R_. tigrina and three in l_3__u_f_o_ stomaticus by Sharma and Dhindsa (1955) . Burgos and Ladman (1957) , briefly described two types of spermato- gonia in R. pipiens. Glass and Rugh (1944) and Rugh (1951) have briefly described, also in R. pipiens, a single type of spermatogonium which appears to be similar to the Primary spermtocytes described by the other authors mentioned. The photomicrographs of Glass .and Rugh are of such low magnification that they are most difficult to interpret . In R. pipiens and _R_. clamitans testes, at least three types of spermatogonia are present. Figure la is a light micrograph of what is called in this paper type 1 Primary spermatogonium. Figure 1b represents type 2 Primary spermatogonia. Type 2 spermatogonia are matura- tion products of type 1 with no intervening mitosis. The 18 19 Figure l. Spermatogonial types-light micrographs. a) R. pipiens b) R. clamitans c) R, pipiens Note the type 1primary spermatogonium (Tl), groups of spermatocytes (Se), type 2 primary spermatogonia (T2) separated from each other by follicle cells (Fe) and a portion of a cluster of secondary spermatogonia (2nd). x 2,000. ary her . cl. 5"? 1' I "' 7 u ":E. “a. .3»... _ .513}. A smgie : ' ‘ .J. n. ‘1‘“ n? fltiflo :‘OVOSOVOJ 550...“. . I. AI‘ :1”: tapes of the a """ Int 3 “ 'eeés “U u 5 EMS size basis c‘ s L " ‘h 4A e ' : ~~ge ‘fien'Q‘ R: ‘..\'u was classes c ‘ \ 0“ :In‘a A“ . H U \B‘bmy..b ‘ ‘ . ~."R. ‘. 1:“ ' ' .T-‘ode‘us' a“ u- 5.. 1 ‘5! also e... K I' , :‘QQC I co.::.‘Y‘ H “‘5 Eye: 21 older literature grouped these two types together (Champy, 1913). A single type of secondary spermatogonium which is a division product of type 2 primary spermatogonia is also present (Figure 1c). These cells occur in clusters while both types of the primary spermatogonia appear as individ- ual cells not directly associated with other germinal cells. 7 In mammals spermatogonial typing is usually done on the basis of size and nuclear structure. This has led to the identification at the light microscopic level of various classes of the two main spermatogonial types, A and B (Clermont and Leblond, 1959 and Roosen-Runge, 1962). Amphibians, although not studied in as much detail as mammals, also evince nuclear morphology that aids in classifying spermatogonia. Three types of spermatogonia are also visible at the ultrastructural level in R. pipiens and R, clamitans testes. However no definite ultrastructural variations are evident to distinguish between the spermatogonia of the specific types in the two species, so the following description will suffice for both. ‘ Iypg_1 Primary Spermatogonia--Figure 2 shows a type 1.primary spermatogonium. Primary spermatogonia are relatively large cells (10 to 15 pm in diameter) and have a circular-appearance in sections. Each primary spermato- goniumlis isolated from the rest of the germinal cells by 22 Figure 2. Type 1 primary spermatogonia, R. pipiens. Note the nucleus (N), nucleoli (Nu), mitochondria (M) and the follicle cells (Fc) surrounding the spermatogonium. x 10,200. 23 .u... ..l. ., .11. - . ‘ s :on #5.“ j“: .V...:.& .e a 1 ' 'F‘ .n D! 3' ‘ .40.:0 Hoe :1: I ‘ q ‘ P“ : ' F.1UF'3‘ a... tube-d. n -- .I-IIO O" I! :0 I:::. b. 0| 6 0.: an. en". :5 maid. v:..‘ C l ' u UAI.:’:.e I. W“." - I ‘ I-n . . - — F ‘ _,.:L .C. r ,. Olive-.1 . a " m Nee b I a ‘I.". t ' pp... he“. ‘ 5V‘\ g. ' c . ..‘-A""F‘ : fivvvnvao‘- .a I 'V‘.v..A ‘ ‘5 w nu..:‘ e.'cu in '3‘:-.~ e ~“:|I‘" “Av . ‘ ' ‘98. I A ‘- “Q. Q ~ ...‘I ‘0“ n... ‘ ‘5 I It I Q‘. s G' a It... =cah ‘ 'Vh \‘ “"3; In“ : ‘ "o 1 :5.‘§ u“ g "‘ Ah.l ‘. " " ‘v e. Q ‘Q R.A q ' "I.“ :Hv' § 0“ : s~~- I.|:‘ I -C ‘- \.e inn“. “4» . C. ‘u 5 n \ Q‘ 3145 A ‘ ‘CI w... ‘0 u‘ ‘ | "-5 ' . J M. “.5 24 being completely surrounded by follicle cells. The nuclei are approximately circular in sections. The nuclear membrane is smooth and double walled. There are at least two nucleoli (Sharma and Sekhri, 1955) present. The nucleoplasm is finely granular with occasional clumps cfi’moderately dense chromatin in association with the nuclear membrane. The cytoplasm has many free ribosomes and a small ammunt of rough endoplasmic recticulum (Figure 3a). The autochondria are circular to oblong in sections and dis- tributed evenly throughout the cytoplasm. There is a tendency, however, for a small amount of mitochondrial Cflnmping, similar to that observed by Nicander and Ploem (1969A) in rabbit type A spermatogonia. The mitochondrial cuistae-are dense and highly branched. The intercristal matrix has little eleCtron density. Membrane scrolls are Often seen (Figure 3b) in association with the mito- Chondria. In fact, what appears to be the beginning of Scroll formation is often seen as a blebbing of the outer mitochondrial membrane (Figures 3a and b). Small dense bodies are seen in the cytoplasm close t° the nuclear membrane (Figures 3a and b). They have the Same density and appearance as the chromatin condensa- ti°n8 in the nucleus and in some cases are associated with m'1t<-‘*<=hondria or membrane scrolls. (For further discussion ind 25 Figure 3. Portions of type 1 primary spermatogonia. a) R. pipiens x 33,000. b) R. clamitans. x 33,000. Note the nuclei (N), rough endoplasmic recticulum (RER), ribosomes (R), membrane scrolls (S) and mitochondrial blebbing (MV). Arrows indicate the cytOplasmic dense bodies. A portion of surrounding follicle cells (FC) is alsoivisible. , . no one u I“ isnse by: Nwzba'uhn; a.\‘ accuvvd‘vn. o 4 "III a .:e al:.:. :o-n: ’ a II... ‘ a“ R“ : on but '1 u fl‘f‘r' '- "‘" "v weave 'II. . ' ‘1, YIQ‘I “"10 ““‘ .9 IF I Q .Je var" .31 in .§. . ‘ - F! ‘0‘. c 1 U: ‘ . ": :‘Rnk‘; e V3” 04 u u . bo.‘ 27 of the dense bodies see the section on type 2 primary spermatogonia.) The characteristics of type 1 primary spermato- gonia are almost identical to the rat type A spermatogonia first described at the ultrastructural level by Watson (1952). Similar characteristics are also found in the amuse (Gardner and Holyoke, 1964), rabbit (Nicander and Idfiem, 1969A), and human (Tres and Solari, 1968 and Andre, 1962). Brokelmann (1964) showed cells of similar nmrphology, both at the light and electron microscopic levels in R. temporaria. Typg_2_Primary Spermatogonia--Type 2 primary Spermatogonia arise from type 1 primary spermatogonia. However, this change in cell type does not take place after mitosis for type 2 cells still appear individually (i.e.: not in clusters). Champy (1913) regarded type 2 SPermatogonia as mature "gonia 1." The most characteristic feature of type 2 primary sPermatogonia are the polymorphic nuclei (Figure 4). The nuClei appear either as crescent-shaped, reniform, dumb- be11 shaped or multilobed. Polymorphic nuclei are charaoteristic of spermatogonia of many amphibian species, a1though much-variation in the degree of lobing is evident (Champy, 1913). Type 3 spermatogonia are much larger than type 1, Itleasuring about 20 to 25 um in diameter (Figure 6). .' ‘I ‘- -T' Figure 4. Type 2 primary spermatogonium, R. clamitans. Note the lobed nucleus (N), two nucleoli (Nu), clumped ribosomes (R), cytoplasmic dense bodies (DB), membranous scrolls (S) and mitochondria x 10,200. (M). 28 29 EDI-l5 (1963) ‘3 .nu::. ‘ ‘. oily-VF “A "‘ uuo:.u 5‘ .h‘n I ' a .. I-Ifll "A”? ‘ .0 card Vv‘. ‘ I I ‘ at. .. ‘IR' ‘ .u m finvbeas I ' . .. .nnvvx' ‘ "‘ ‘ “’1 "HI". ~.¢.U . I . et‘ Eyes 4 a" ‘- as! a ‘ I .1628 I zate.:;:.er.t as I :‘n “‘ Q.» I v, I do. '- ~ .u 1:“- v on ' "'0 nee "7‘ 30 Monesi (1965) has reported a very high synthesis of protein and RNA in type A mammalian spermatogonia which, if such occurs in amphibian spermatogonia, may account for the increase in size. Apparently the nuclei become polymorphic and then the cells increase in size (compare Figures 4 and 6) . There is an increase in the membraneous scroll development associated with the mitochondria (Figures 4 and 5). The mitochondria are still round to oblong in section and the matrix is clear. There is a tendency now for the cristae to become parallel with less branching. Instead of being randomly distributed the mitochondria (are now clumped around the dense granular material. In some cases there is a partial polarization of the mito- chondria to one‘portion of the cell (Figure 6) . Aggregation of mitochondria in clusters around dense material has been seen in growing oocytes of mammals (Adams and Hertwig, 1964; Hope, 1965; Odor, 1965 and “haakley, 1966) in some fishes (Zahnd and Porte, 1966) and 8Qme amphibians (Kessel, 1966, and Clerot, 1968) . Nicander a11d Ploem (1969A) were the first to describe dense inter- Initochondrial material in mammalian type A spermatogonia. :tzhas been described at the ultrastructural level in Inammalian spermatocytes (Andre, 1962; Eddy, 1969; Nicander ‘and Ploem, 19693 and Fawcett and Philips, 1967) and in Spenmatocytes of three species of Rana (Clerot, 1968). 31 Figure 5. Portions of type 2 primary spermatogonia. a) 3, clamitans. x 25,000. b) 3. pipiens. x 33,000. Note dense bodies (DB) in close association with the nucleus (N) and the associated mitochondria. Arrows in 5a indicate membranous scroll-like bodies that may be forming mito- chondria. In 5b the arrows indicate the close association of the dense bodies with the mitochondria. .Membranous scrolls (S) are also seen. I. -__4 - __ --‘ «Ln-EMA? _F. _ - ‘ w ‘ . . Mrg.‘v'-'_ . . . ... -_ a“ ‘. ' .I ' ' o . . 33 Figure 6. Type 2 primary Spermatogonia, 5. pipiens. Note the intermitochondrial dense bodies (DB) with the associated mitochondria (M), follicle cell (Fc) membranous scrolls (S) and the nucleus (N) and nuCleolus (Nu) of the spermatogonium. x 12,600. 34 ”3A,: .0. .V Q .v-o-w- AI an H n—' cod“ ‘0.“ L‘szazce. F ' ‘ Al pun A2, A‘ u ~OU.:“ y. "~~:-~' an 4 “V0“..v'.‘ i ‘l.:”’°+ ."_ l ~'~°""5 run 0 ‘ II..:. {'1' f" "N 0. vu- '-...'. V 'iouaol::e: + b . v... ":1:- our... '3 . I m,‘. . ‘ nhvu‘v‘. .n . T“ :‘e ... fin. 0 I"' ‘. :..‘R;. . ‘==.'.:.‘ 0— U.’ ‘5 =24 .. , I“ =V e A I.- “a I I . Q .i‘ ~ .."= . :V‘n ‘0‘ ‘ ~A.- ‘ "":‘: A: 5.... 35 There has been much debate about the origin, function and final fate of the dense intermitochonrial substance. Kessel (1966) suggests that the material is of nuclear origin and that it may function in transfer of information to the mitochondria. Zahnd and Porte (1966) interpret the dense material as ribonucleoprotein trans- ported from nucleoli to the cytoplasm. André (1963) considered the dense material to be aggregations of ribosomes. Odor (1965) suggested that this material may function in is r_m_v_o_ mitochondrial formation. The in- complete outer mitochondrial membrane often seen in association with the dense material (Kessel, 1966; Odor, 1965 and André, 1962) (also see Figure Sb) was thought to add some degree of credence to this supposition. However Fawcett and Phillips (1967) and Nicander and Ploem (1969A) Suggest that the incomplete membrane is due to obliquity 0f section. Fawcett e3 a_l_. (1970) suggest that the Chromatoid body may be a product of mitochondrial synthetic aetivity. In mammalian species (Eddy, 1969; Nicander and Plb'em, 1969B, and Fawcett'and Phillips, 1967) , the forma- tion of the chromatoid body has been described as a coalescence ofthe intermitochondrial dense material. The cihromatoid body is then divided amongst the spermatids and fOrms the major component of the annulus. Recently Fawcett at it: (1970) have shown in a variety of mammalian 1"“-— FILES :13". I n: ‘0‘” -\ u a In tunic! .....‘4. .6," LOOd "5". "Of " \ U‘ u . Q ‘1 "can a a 3...:: ,3“ .. II .‘:—n‘+ ‘ U.v..4y.‘., ‘ ' a 0"":093r A " " '5 V. ‘ 1 I:O:'.: ’ ' ~I'..‘. ..‘ ‘GC: "‘1' 0.4,). "o... h . ‘. d ‘3‘} Q _ 5.. :1... u: '18 .g‘ Q N:;a =ua~ 5 iv. ‘ U I.” . ‘ ‘ A. Nuvv..v..c7 ‘n \ ‘3 ‘ ‘v ‘. «v. "-E I 0.. "‘ o ‘Q -, yvvnvrfl‘ .l 36 species that the chromatoid body does not contribute to the annulus but seems to gradually disappear as the annulus migrates caudally. Cytochemical studies on the chromatoid body have suggested the presence of ribonucleOprotein (Daoust and Clermont, 1955; Sud, 1961 and Fawcett and Phillips, 1967) . However Clerot (1968) found that the intermitochondrial material in oocytes, and spermatocyte of three species of 322.9. is sensitive to protease and not RNase. In mammals (Eddy, 1969 and 1970) the chromatoid body. is‘resistant to both RNase and proteolytic enzymes. Since amphibian spermatozoa do not have an annulus (see section entitled "Spermiogenesis") , another function would have to be suggested for the chromatoid body in these species. It is (indeed possible that the inter- mitochondrial material found in amphibian germinal cells is not the same type of material as that found in mammals. Although it seems improbable that the dense inter- mitochondrial material functions directly in d_e_ £919 mitochondria-l formation, it is still possible in amphibians at least, that André's (1962) other suggestion, that the intermitochondrial material may stimulate growth and/or, diVision of mitochondria, is correct. There is a definite increase in the number of mitochondria from type 1 to 1imbe 2 primary spermatogonia (compare Figure 2 to Figures 4 and 6) . This increase of mitochondria takes place in zmsmc . Il.. I Oil'- ‘9. 4::v ibtuvtl v ‘. .- ima,l 22:57.9 SCI q u anus. ...:v I ' Q n.- .‘ ‘ --~F'. I ~°“. ~c.-.c fififiei .:.:" Q“ 1 d ‘u‘..‘. . u :I- .‘.AR".A‘ ~‘ N'V‘qu, u' l: I C” v- 0... ‘V‘e..e ' e...- .g hie: ‘L a "e ‘- = VHA“. d i V Q V‘s. . "H: 1: U ‘ F 5‘ sn.\. .‘ I .1 n c ““‘e‘l ‘5 Lb ." n u‘;l ‘L ».;:‘ ‘ : ai:§ .I. 5 u. t F n‘. '1‘: 5““ 37 mammals in the growing primary spermatocyte (Johnson and Hammond, 1963) , where the intermitochondrial material is best demonstrated (Fawcett and Phillips, 1967 and Nicander and P16em, 1969A) . Figure 5 shows the association of membrane scrolls in association with dense material. The scrolls may be formed by blebbing of the outer mitochon- drial membranes (Figure 3a and b). Figure 5b shows what may be the formation of new mitochondria frOm scroll-like material. It is equally possible that the membrane scrolls and mitochondrial blebbing may be fixation artifacts. These blebs and scrolls may also be the first indication of cell degeneration. Many spermatogonia do not develop further than this stage (Lofts, 1964 and Champy, 1913) . Some type A spermatogonia in mammals reproduce periodically to replace those spermatogonia which develop into spermatocytes (Roosen-Runge, 1962; Clermont, 1967 and Clermont and Bustos-Obregon, 1968) . Although primary sPermatogonia of g. clamitans and g. pipiens are similar in structure to type A stem cells of mammals, it is not known if they are functionally the same. Without triated 1"hymidine-labeling experiments one can not be sure. How- EVer, it is. assumed (Sharma and Sekhri, 1955 and Champy: 191.3) that they do function as stem or reserve cells. The fact that primaryspermatogonia are individual cells also adds some support to this idea. ll wua kn III-lb .Icob U a. H. c i t :v u 'Qfiy mo' 'v.n 0 cont... .- AJ a A I -v-o‘oaoaga v‘ it:n.. f‘fi“ my! ‘. .‘ ‘0 I-cl..~ ;;" ~""V hot. 1.. 0!! Gk.- . ~ ~: 'V boost... ...-."7 FA ."---‘-; 5...: . (I) (D ( ) V... ‘ I h ' " “we 1 I \. V 'n(. i ”<1 19: I 3;. Ike ~“F. can ‘ p -‘ u..-‘. .I .‘h‘ “='r“~l ‘ ‘r. "v ‘V. i A gas“, I‘ I‘- s‘s 0‘ a .I N’. ‘ ’ “\s‘wn "» ”C V § 0.1 “""A‘ "‘9 1.- I ‘J 38 Secondary_Spermatogonia--Secondary spermatogonia arise by mitotic division of type 2 primary spermatogonia. Early workers (see Champy, 1913) suggested amitosis as a mechanism of division for type 2 primary spermatogonia. Champy (1913) and Sharma and Sekhri (1955) described mitotic division of type 2 primary spermatogonia. Accord- ing to Champy (1913) amitotic division of the type 2 primary spermatogonia leads to cellular degeneration. Secondary spermatogonia are found in clusters. The size of the individual cells and the number per cluster depends on the number of divisions which has occurred from the primary spermatogonia. Figure 7 shows a portion of a cluster of secondary Spermatogonia. Each cell in the cluster is separated from its neighboring germ cell by only its plasma membrane. The whole cluster of secondary spermatogonia is surrounded by follicle cells. The nuclei have an increased number of thickenings that have'been described as chromatin "flakes" (Sharma and Sekhri, 1955) . The rest of the nucleoplasm still has the fine granular appearance. Sharma and Sekhri (1955) and Champy (1913) show similar nuclear features in their descriptions of secondary spermatogonia. These nuclear features of amphibian secondary spermatogonia are charac- teristic of mammalian type B spermatogonia (Gardner and Halyoke, 1964 and Nicanderrand PléSem, 1969A) . 39 Figure 7. Secondary spermatogonia, R. pipiens. Note the nuclei (N) of the six secondary spermatogonia, intermitochondrial dense bodies (DB) surrounded by mitochondria (M), ribosomes (R) and the plasma membranes of the spermato- gonia (PM). A portion of the surrounding follicle cell (PC) is also visible. x 10,200. 40 ’3- .5. . QQInsfl RF“: “gm-u bush- 1 , at. "F. 1‘. loavul .g. - . "'Fv‘nn‘. L‘:Hvi ="::':'“ES 2...". lug nipV"" HR ""“ cad! . "L. l ' :A...‘ w'UUh-c . :.:. "A“. I UM... C III.“ .. _. ‘. 3".- ‘ \ 5 ‘II "V~.'.“ 1 UVH‘ a: a h I Q I v a a ‘ N.. f‘. 5“. i.‘ u“ ~~“‘.' I " h ‘1‘ ‘ U i . .I Cl. \ * a :‘~.'~ \‘ v.‘ '— H ‘L ‘ .\--~ . '- 41 The cytoplasm of secondary spermatogonia has changed considerably. There is a reduced amount of cyto- plasm. The number of mitochondria are reduced. The dense intermitochondrial material is still present but the rumbranous scrolls are no longer evident. From all appearancesmitochondrial multiplication is no longer occurring. There is however still a large amount of free ribosomes. No intercellular bridges have been observed between spermatogonia of -any stage in R. clamitans or _R_. pipiens testes. Nicander and Ploem (1969A) observed. bridges between type B spermatogonia in the rabbit. However, Gardner and Holyoke (1964) and Fawcett (1961) suggest that the last spermatogonial division is incomplete thereby giving rise to conjoined primary spermatocytes. No crystalloid bodies have been found in the sDermatogonia. To the present, crystalloid bodies have only been observed in human and, posSibly, chicken sper- matOgonia (Nagano, 1969). Meiosis In most sexually reproducing organisms the doubling of the chromosome number at fertilization is offset by I‘eclucing the chromosome number of the gametes to half their diploid value during a stage in their development . This Change is brought about by a single chromosome duplication (Fly-.1 A ;.I‘.ll;~. kfi' nooi‘bi U: I u :¢;v~u30r\f It U'VOO~UJ‘ . I II! "Q. hid “ SAN cw .. vu . ‘ ' z“ an” ,: I .""" 'Vdcu . _ u .ll.v.~'. ‘ I ~.I..Ia‘ | 2 i":' AIM a.“ 'V: g. I :u‘. . . § I o-az‘.2::~ ""va: Qv. '.' . "Vle c.‘ | .Fn . o 'c. \u. ‘ a. -: I. g.‘ A54” v-‘. Q E - '. .II ‘og “i.- i U ‘0 I “Q l 'A u eroc ‘ .. I a a. 2 . 42 followed by two successive divisions. The process is called meiosis and occurs during the spermatocytic stages of spermatogenesis. Chromosomal movements and behavior during meiosis were well documented by the early light microscopists (see Wilson, 1925) . However, subtle changes in the cyto- plasm could not be detected. At the ultrastructural level individual chromosomes are either too diffuse to be seen or, when compacted, need to be serially sectioned for full visualization (Solari and Tres, 1970) . At the electron microscopic level cytoplasmic variations can be determined. However, even in well characterized mammalian cells, descriptions of; morphological changes during meiosis are scarce. Nicander and Ploem (1969A) described the stages 0f meiotic prophase in rabbit showing micrographs of whole Cells-but only “in the leptotene and pachytene stages. The only micrographs of the ultrastructure of amphibian sPeeratocytes were (published by Clerot (1967) . He was bi'leically interested in the dense intermitochondrial material and not in. the spermatocyte p_e_r_ 1e . In this study an attempt was made at the electron microscopic level to classify the stages of the first meiotic prophase by relating the "typical" light micro- 8capic observations of chromosome behavior in meiosis with the observations of Rugh (1951); Sharma and Sekhri (1955) and Champy (1913) on amphibian spermatogenesis. are- _'_ . hop-I - a a ‘ - "with cod I j.::j.:.a‘e was - a . “V PFC! 0.0 H» p... u! ::.-.‘ A“ "' "'UoVu "2.35 are ‘l- ::‘AI~‘, nunv..ua‘ F; ‘0‘ ya...“ . o“! [J . o (It 0 1 t): . I ‘I try 43 No distinguishing differences between the two species in the ultrastructural aspects of the first meiotic guophase was noted. The following description will suffice for both. Figure 8 shows a portion of a cyst of interphase guimary spermatocytes. The nuclei are circular to oblong in section with somewhat irregular outlines. Chromatin clumps are seen on the periphery of the nucleus with occasional condensations in the nucleoplasm. Two nucleoli are seen in some sections. Two nucleoli have been de- scribed in the primary spermatocytes of R, tigrina (Sharma and Sekhri, 1955). The mitochondria are still clumped around dense Haterial. Intercellular bridges are seen, which, according to Fawcett (1961) and Gardner and Holyoke (1964), are an aid in distinguishing primary spermatocytes from late Spermatogonia. According to the above authors, inter- cellular bridges are formed at the last spermatogonial division, just prior to the transformation into spermato- cytes. Rounded, swollen (active?) Golgi elements are Visible in addition to the more flattened type. This is the first time Golgi elements are seen. They have been described, however, in mammalian spermatogonia (Nicander and Ploem, 1969A) and at the light microsc0pic level (Sharma and Sekhri, 1955 and Sharma and Dhindsa, 1955) in 44 Figure 8. Primary spermatocytes-interphase, R. pipiens. Note the mitochondria (M) still clumped around dense material, Golgi elements (GE) and inter— cellular bridge (IB) between spermatocytes. x 6,000. m-“ Il‘ . . . .un-h\:l P I “MOOO‘OO u 0 h 52.1! to t . fit a a. O 25:36: 9. :.:;s are r I u U- I: ‘."\-A‘ .“~ V“. tinny. .: Jinar Q n. a ‘A v . Q“ H e. S ‘ o ‘5'. I‘ u I . ”ou‘r‘: c I‘.‘ UN. Nubia . 0 ¥. ‘ .5 £7. M;:3 .u a. b L‘ .C I': J‘Cfih so 46 amphibian spermatogonia. The rest of the cytoplasm is similar to that seen in the secondary spermatogonia. Leptotene stage primary spermatocytes are shown in Figure 9. Most of the peripherally located chromatin clumps are'no longer present. Instead, a large number of fine chromosome threads are visible in the internal nuclear area. It is thought that Figure 9 is-a cross section through the so-called bouquet arrangement. The nucleoplasm is granular and the nucleoli are visible. In the cytoplasm the Golgi elements are circular in section with large vacuoles. Few distinct lamellar structures are associated with them. Clusters of free ribosomes fill the cytOplasm and a small amount of RER and SER are visible (Figure 10). Mitochondria are oblong in shape with distinct cristae. No membranous 'scrolls or mitochondrial blebbings are seen. Mitochondrial clumping isstill visible. No definite relationship of the Golgi elements, centrioles and mito- chondria, as described by Sharma and Sekhri (1955) , could be distinguished. BY the zygotene stage the chromosomes are pairing. Notice the thicker, reduced number of chromosome threads in Figure 11. The nucleus is‘still oblong in section with an irre91.11ar outline. Solari (1969) finds synaptinemal complexes in mammalian spermatocytes of the zygotene stage. Mitochondrial clusters are breaking down. Accord- mg ‘10 Nicander and Ploem (1969A) this occurs in the late 47 Figure 9. Primary spermatocytes-leptotene, R. clamitans. Note the chromosome threads in the nuclei (N) the mitochondrial clumps and Golgi-elements (GE). x 5,000. Figure 10. Cytoplasm of primary spermatocyte-leptotene R. clamitans. Note the rough (RER) and smooth (SER) endoplasmic reticuli, portion of an intercellular bridge (IB), Golgi elements (GE), clumped ribosomes-and dense intermitochondrial material (DB). Arrow indicates intramitochondrial material. x 26,100. 48 I a}! I; );v 49 Figure 11. Primary spermatocytes-zygotene, R. clamitans. Note the thickened chromosomes (C), mitochondria (M) and Golgi-elements (GE). x 10,200. Figure 12. Cytoplasm of primary spermatocyte-zygotene, R. clamitans. Note the smooth endoplasmic reticulum (SER), clumped or isolated ribosomes (R). Arrow indicates dense intramitochondrial material. X 22,2005 on, évjssafifl .Q‘-‘ ‘ 0"": ‘ _SL ell'lnbllfl 9‘ Acu‘ .to‘ 3' n I g” In ‘nr-v -: “IV .Uoofl‘u ' 0- . "‘ ‘vuq .v.‘ u. .‘ u.“ . “‘ t'LW-e FA v.0..4 'Vy‘v.t ‘ \"I n ‘ ‘6‘ j C '. "Cy . I 3‘ ‘ O .“Ucuc 51 pachytene stage in rabbit spermatocytes. The ribosomes are no longer arranged in clusters but are spread individ- ually throughout, the cytoplasm. Cisternae of smooth endoplasmic reticulum are becoming more numerous. Little or no RER is visible (Figure 12) . Occasionally micro- tubules can be seen. The next stage is called the pachytene stage and is the longest period ofprophase (Swierstra and Foote, 1963) . In the early pachytene nuclei of R. clamitans spermatocytes the chromosomes appear as thickened struc- tures one less dense nucleoplasmic background (Figure 13a). Synaptinemal complexes are seen for the first time. The lateral components of the complexes are separated by a 90 nm space and the central component is about 1.5 nm wide (Figure 13b) . Similar measurements were given by 501311 (1969) for synaptinemal complexes found in mice spermatocytes . The Golgi elements have lost any similarity to the characteristic flattened form. The mitochondria appear smaller than in previous stages. A definite chromatoid- 1ike body is present and the mitochondria are spread through out the cytoplasm. Occasionally some dense inter- mitochondrial material is still visible. By late pachytene (Figure 14a) the chromosomes have thiclcened to suCh a degree that they fill the whole nucleus (also see Rugh, 1951) . The nuclei appear circular 52 Li Figure 13. Primary spermatocytes-early pachtene. a) R. pipiens. x 6,000. b) R. clamitans. x 66,000. Note the Golgi elements (GE), chromatoid-like body (Cb), synaptinemal complexes (Sp) and nucleolus (Nu). ..,‘.‘A' 0' " . I. i. 4.. d. . |‘ F 0'. "0:! ‘ e. . In: '0'.“ The 1:;- .‘04'A :uut o‘er, I Last, are ~ I "...a.. .v : ::ovcoa‘ U- ."'i‘:.‘u ‘1 Miu.‘ b... I 0‘:--’ A: v - . It... it. . *2'ua. . ""‘cu.“’ c ‘l " ”5". f!- 0- hcdvbvh'é ‘l..‘- a ‘0“: of j u .’A'Fq. u Carynaa . . I:Og~"“‘ ”vb-u .: . O I ~.: SEE ~‘. a. n i ‘I ""‘I3.“ i...‘ I “AA" '5' \h-_ 0 U». 54 in section with a smooth outline. Synaptinemal complexes are visible. The cytoplasm has changed considerably (Figure 14b). Mitochondria, although about the same length, are half-the width of those seen in early prophase. Vesicular SER is very abundant and occasionally large liquid-filled vacuoles are seen. No RER is visible. The number of ribosomes appears to have decreased and those remaining are again arranged in clusters. The variations in ribosome arrangements may indicate a variation in the amount of protein synthesis during the different stages of prophase, as described by Monesi (1965) in mice sper- matocytes. Dense bodies similar to thOse seen in sperma- tids (see page 80) are distributed throughout the cytoplasm. The number of microtubules has increased and they seem to have their origin or terminatiOn in associa- tion with the centrioles (Figure 14c). The cells at this stage are about 10 pm in diameter with a nuclear diameter of approximately 7 um. The size of an interphase nucleus measures about 5 pm. During the diplotene stage one set of sister chrmmatids begin to separate from the other pair. Wilson (1925) states that spermatocyte nuclei of various species "recede or deconcentrate" in interphase nuclei in early diplotene. This sort of change, according to Wilson. is correlated with the processes of cytoplasmic growth; in 55 Figure 14. Primary spermatocytes-late pachytene, R. clamitans. a) x 5,000. b) x 15,300. Note the synaptinemal complexes (Sp) in the dense nuclei (N), smooth endoplasmic reticulum (SER), mitochondria (M), clumped ribosomes (R) and "fluid filled" vacuoles (VF). -‘ ‘ Ar r"- "—1 _‘ I .Auaya OI a I:nvo 15 L- l p. A y no ()1 a — llvov a Apia-v II I 5 I! A vial-u3fi u ‘ , :0: “~' ' UV cub. I "DI. ' 34...: t v»! a I In a. ' '::e A! a l l.. ' \ ‘l‘ ‘, u,“ e: I I... . 57 general the longer the growth period the greater the nuclear diffusion. However Solari (1969) states that condensation of chromatin material in the mouse spermato- cyte increases up to early pachytene, then slow "unconden- Sation" takes place up to diplotene. The diplotene nuclei of R. clamitans spermatocytes (Figure 15) are less dense than those of the late pachytene Stage. Synaptinemal complexes are still visible. The Cytoplasm is identical to that described for the late pachytene stage. The cell has increased in size to about 12 to 14 um in diameter. No definite sex vesicles, heterochromatic sex pair, were distinguished at any of the stages of meiotic prophase studied. The remaining stages of the first division c0uld not be identified. Secondary spermatocytes arise from the first Ineiotic division. In R. pipiens they are said to be much Smaller in size than primary spermatocytes (Burgos and ILadman, 1957 and Rugh, 1951). Burgos and Ladman (1957) <3escribed the nuclei of secondary spermatocytes as having jperipherally located chromatin clumps. Rugh (1951) de- scribed the nuc1ei as uniformly stained. This of course may reflect different stages of the second division. Rugh also states that these cells are located towards the lumen and that the cytoplasm may be tapered at one end. 58 Figure 14c. Cytoplasm of primary spermatocyte-late pachytene, R. clamitans. Note the centriole (C) with the associated microtubules (MT) and a Golgi element (GE). x 26,100. K! “'EIT'“ Figure 15. Primary spermatocyte-diplotene, R. clamitans. x 10,200. '1‘ v Al - _ , (C r . . M . u v. .. «J a. {,1 r1. .. 1.. . 7. I. . w . ! one a Rhos-n on: :bviuu Q (In nng ‘ u: “iv. . (I) (h ' ‘ ’2""“ tr "OOJi' Us 0 | I n... .“ "'0:¢Vu 0 22:5 15 0.: c. F s...”- ' n . ': :5: f‘ by. l a"; 60 In 5, tigrina, Sharma and Sekhri (1955) described the secondary spermatocytes as having homogeneously stain- ing nuclei. The mitochondria appear as "tiny particles." Secondary spermatocytes have a short interphase period, that is, they rapidly go into the second meiotic division. In some insects, for example, the second meiotic division immediately follows the first so that there is no interphase and the te10phase chromosomes of the first division are directly transferred into prometaphase of the second (Sharma and Parshad, 1955). No positive identification of secondary spermato- cytes could be made in this study. §permiogenesis Spermatids arise from secondary spermatocytes as a result of the second meiotic division. The series of developmental events by which sper- matids are transformed into mature spermatozoa is called spermiogenesis. In mammals spermiogenesis can be divided into four general stages that are defined by the deve10p- ment of the acrosome (Bloom and Fawcett, 1968). More specifically, however, 19 stages were identified by‘ Leblond and Clermont (1952). In 5. pipiens and 5. clamitans, spermiogenesis varies considerably from the precess in mammals so no attempt will be made to fit amphibian spermiogenesis into this well defined system. - u an ":7 . LIEV‘..' in 3...}; a5 u vvav \- 0] :h‘ ‘. ‘. V. V a b no .efioavé I'VVFT":J w h'volvu Ey'beau 'vnA. 14.1,. ‘ I.’"Qbo.' :.....,e, .‘Icqvfl I..' I- OF a “V hi I 5- t a, . I H." n . "l . bee: 2) ( .5‘1r4:‘ . . Q _- ‘ yu‘h'Va “,3: a '06“ .F’I. U. 5 ‘l .A 503‘s . ‘M 5“. ‘ § \ ‘ 9 ~. 61 However, during development of the mature sperm, in most species at least, three major phases can be described: 1) Formation of the acrosome. In acrosome-bearing flag-ellated spermatozoa the Golgi element is usually involved with acrosome formation. That this association may be a universal occurrence was first suggested by Bowen (1920). The acrosome usually a membrane-bound bag-like structure, carries the enzymes needed to dissolve or rupture egg coverings so penetration can take place (Tyler, 1939; Berg, 1950; Wada et_al., 1956; Dan, 1967 and Srivastava gt_§l., 1965). Because of their similar mode of formation the acrosome has been called a specialized lysosome and indeed some of the enzymes found in lysosomes have been found in acrosomes (Anderson, 1968). 2) Condensation and elongation 9£_the nuclear material. During spermiogenesis in the chicken the 3 to about 2 um3 with nuclear volume decreases from 110 pm a change in the axial ratio from 1:1 to 22:1 (McIntosh and Porter, 1967). There is thus a substantial condensa- tion or elongation of the nucleus. The elongation process in chicken spermatids has been associated with elaborate helical microtubular formations. The—manchette, a well known microtubular structure in mammalian spermatids (Burgos and Fawcett, 1955), is also thought to function in Sperm shaping. Microtubular formations during sperma- tid elongation have been described in a wide variety of l ‘ i!” 333.55 uw ":a“‘: ‘“ ' ~ I- uuvv nu \HUU IV.“ A; I‘IA '0'“ it I‘IH itSPEClEE ‘."‘H.:I'A a :~""‘O:D 3h"-~ “1" a“.... M“. o ‘ I lac-'3' I c .. .._ne-‘ ‘ on. ....e:s ( ‘u.,.. .1. V \.,:‘S p A '.:L.=‘: .. ‘ an. t, "t. c u in. “ t...‘ ~G 62 species including the earthworm (Anderson gt_§l., 1967), insects (Robison, 1966; Philips, 1966; Kessel, 1966 and 1967), and mammals and birds (Nicander, 1967). Condensa- tion of nuclear material occurs in various ways in differ- ent species, varying from clumping of the chromatin to eventual fusion of condensing parallel thread-like structures (Fawcett, 1955). Karyolymph is eliminated during nuclear cOndensations and is expelled in clear membrane-bound pockets from the posterior portion of the nucleus (Andre, 1963; Franklin, 1968). 3) Midfipiece formation. The centrioles are first seen at the cell periphery in the early spermatid. From what will be the distal centriole the axial filament begins to grow. Axial filament formation is in itself not a universal criterion for the spermatid stage. Meves (see Wilson, 1925) has described four axial filaments in the primary spermatocytes of the butterfly Pygaera. The centrioles with the elongating axial filament begin to migrate to a position close~to the nucleus. Golgi elements are usually associated with the centrioles at this stage. The Golgi element and the centrioles then separate and take positions at opposite portions of the nucleus. Mitochondria become associated with the centrioles for mid- piece formation and the Golgi element functions in acrosome formation. ' I I A ”'3; Fl“: pan-v Uni O .433. V .1... n V ’3. ‘ O M «A u.- and PI a ' : :“ p9 ~- ... U. p. O V: O‘ v‘. ‘. y . l:"~ t "- cl."- ‘ " I 'g' (D (I, 63 Extreme variation occurs between species in these three phases.of spermiogenesis. Detailed accounts of these variations are given by Wilson (1925), and Nath (1956 and 1965). The early events of spermiogenesis in 3. clamitans and 3. pipiens are similar enough on a ultrastructural basis to be described together. Acrosome formation, a later event, is somewhat different and will be separately treated. Of the two published accounts of the ultrastruc- tural events of Amphibian spermiogenesis (Burgos and Fawcett, 1956 and Baker, 1967), both begin their descrip- tions in the mid-spermiogenic stages.‘ An attempt will be made here to desoribe the early events which are common to both species of 3221! as well as the more specific later features of spermiogenesis. Early Spermiogenic Events Characteristic g£_§gth_ Sgecies--Figure 16 shows a portion of two early spermatid Clusters surrounded by follicle cells. Large desmosomes are seen along the apposing follicle cell membranes. The Clusters and their associated follicle cells rest on the - basement membrane which encircles the seminiferous tubule. The nuclei appear spherical in section, averaging about 10 um in length by 6 pm wide. The nucleoplasm is heteroge- neous and clumps of chromatin of various sizes are seen, a 64 E2”- Figure 16. Early spermatid cluster, 3. pipiens. Note the basement membrane, (BM) follicle cell cytoplasm (Fc), nuclei (N) and nucleoli (Nu) of the spermatids, chromatoid-like body (Cb), desmosomes (De) separating adjacent follicle cells and the lumen (L) of the seminiferous‘ tubule. Centriole (C) close to the lumen, just prior to axial filament elongation and associated Golgi element (GE) (see insert) are also visible. x 5,700. Insert x 20,000. 4 65 «Lav-A53: I :'0l .c' U. ..‘...' A III =QMI. .‘ O ':':.. A" ‘ toad-odd ‘ u... H “I I I ""“' be. I 0... V5 "Jill; .D 1:11;}. 'v C ,. ~‘, . I 'V.‘. 6" I "is" He... . I“ -h V V... 66 a characteristic of condensing chromatin (Fawcett, 1958). A single nucleolus is visible in some sections. The cytoplasm is dense and somewhat reduced in relation to the nuclear volume. The mitochondria, spread evenly through out the cytoplasm, are spherical in shape with parallel or branching cristae. The ribosomes are free or clustered in rosettes. In an area of the cytoplasm opposite the basement membrane, centrioles (Figure 16) and Golgi elements (Figure 16, insert) are seen. Figure 16 insert shows a thickened membrane at the extremity of the future distal centriole. This is the first indication of axial filament formation. A small amount of smooth endoplasmic reticular is also visible. As chromatin condensation continues (Figure 17a) the chromatin clumps become larger and the nucleoplasm takes on a filamentous appearance. The nucleolus is still {assent and shows no sign of degeneration. In the cyto- sflesm most of the ribosomes are now in the rosette pattern. Figure 17b is a similar picture of g. pipiens early spermatids showing early axial filament formation (arrow). The condensing chromatin and filamentous nature cm the nucleoplasm‘are most striking. The cytoplasm shows the centrioles with the associated Golgi element, scattered mit00hondria and rosetted ribosomes. The two centrioles in the early spermatid are 1coated close to the periphery of the cell (Figure 16). _F“"'—‘ 67 Figure 17. Early spermatids. a) 5. clamitans. x 15,300. b) 3. pipiens x 17,400. Note the nuclei (N), nucleolus (Nu), ribosomes (R), glycogen particles (G), the Golgi elements (GE) and the proximal (P) and (D) distal centrioles. Arrow indicates area of axial filament formatiOn. -’-. -- ’-_.._-“fl-l_ w. n t g at» ‘~ 5‘-fi{. ’1 8 -. l L-. $13.. sac M-‘vs at b) In“ D b‘ I 1.. I: I‘”“VQ ‘0' «aha.» a ‘ 1 ..‘.A"a O“ Air‘s. . u u a. ”P ‘ I." AM . £112; 5} a, . ! ‘Ao.,‘fl OI way.‘.= {1" II, I" ( 2 A Q.. ' ~. A t“‘~: . \f- § 5 I C!) n-a 69 A small sac or invagination of the cell plasma membrane occurs at the distal portion of the future distal centri- ole (Figure 18a). There is an accumulation of dense material on the inner centriolar portion of the membrane sac. The sac'expands first at the periphery (Figure 18b) and then in the center (Figure 18c) . The sac continues toenlarge (Figure 18d) , forming a cup-like structure bending slightly around the assoCiated centriole. That portion of the membrane sac associated with the centriole is noticeably thicker or. denSer than the remaining por- tions of the membrane. The axial filament grows out into the sac (Figure l8e) and the sac begins to collapse. Finally the sac completelycollapses around the extending axial filament, forming a sleeve-like arrangement (Figure 18f). It canbe noted that the dense material seen earlier has remained at the distal portion of the centriole, in the region where the cell plasma membrane bends back to form the sleeve over the axial filament. The centrioles with the elongating axial filament then migrate to a position close to the nucleus (Figure 189) . The migration process further accentuates the sleeve-like arrangement (of the plasma membrane over the axial filament. The dense material, still seen at the junction of the distal centriole (now, probably, the basal body) and the axial filament, may function as a zone of formation for the Plasma membrane as it enlarges. ,Lw' 70 Figure 18. Axial filament formation. a) 3. pipiens. x 66,000. b) 3. clamitans. x 42,000. 0) 3. pipiens. x 57,000. d) 3, pipiens. x 57,000. e) 5. clamitans. x 33,000. f) 3. pipiens. x 48,500. g) 3. pipiens. x 57,000. Note the distal (D) and proximal (P) centrioles, axial filaments (AF) and Golgi elements (GE). Portion of a spermatid nucleus (N) can be seen in g. The dense material lining the inner aspect of the sperm plasma membrane at the point of axial filament elongation is marked with (-). A6 UNA ( 321:": 0. ol- I Io;v=-e vU‘Q‘U o 01] I ",1'“ FR: I'vo.y..‘, .- . "“‘:u 5‘ undo... ‘ .I I ”.I': "'I.\. 4 .AII . On "I Ne ”v. ‘ f. (D 72 The remaining phases of spermiogenesis in the two species of Rana are significantly different to warrant separate descriptions. Features Characteristic of Rana clamitans Spermiogenesis--Following axial filament formation the nuclei show a tendency to become round in section (Figure 19). Chromatin condensation is continuing but now the nucleoplasm has a fine granular appearance. Vesicles in close association with the nucleus may repre- sent karyolymph and excess nuclear membrane which has been removed from the condensing nucleus. The centrioles have reached their final position at the posterior portion of the nucleus. The nuclear membranes in this region appear thicker and better defined than the remaining membrane system (see Figure 24b). This thickening, also seen in mammalian spermatids, is called the basal plate (Bloom and Fawcett, 1968). Mitochondria, circular in section and with parallel cristae, are beginning to associate with the centrioles for mid-piece formation. The Golgi elements, now a single element instead of multiple as observed in the early spermatid (Figure 20), have migrated to a position roughly-half—way around the nucleus. Microtubules are becoming increasingly apparent (see Figure 23). Two intercellular bridges can be seen connecting three sperma- tids. The bridges are about 1 um in diameter and are similar to those described in other species (Longo and ‘1":- .' :2 73 Figure 19. Cluster of spermatids, 3. clamitans. Note the Golgi elements (arrows), intercellular bridges (IB), granular condensing nuclei (N) centrioles (C) nuclear vesicles (Nv) axial filament (AF) and follicle cell cytoplasm (Fc). x 10,200. Figure 20. Golgi elements in spermatids, g. clamitans. Note the spermatid nuclei (N) and the Golgi elements (GE). x 17,400. .1 o gum" A 5:"... a ' ‘ moccasin] ‘ I neura— n a IDIC'bUOb ‘Illna.a .0 "anti v. on, , lav-o “‘5 we“. I. .1 HM um. ~.' 6 .‘l . ~19, o..~ \b' An. V :fio Q I ‘l ..,:E v I'.‘ ll‘ ‘1: 75 Anderson, 1969 A&B; Fawcett, 1961; Longo and Dornfield, 1967; and Reger, 1963); that is, they are marked by electron dense thickenings on the cytoplasmic side of the plasma membrane. A rib-like structure similar to the connecting piece in mammalian spermatozoa has surrounded the centrioles (see Figure 21a and b). The extension of the proximal centriole into the nucleus may well be an artefact since this is the only time it was observed. The cytoplasm of the surrounding follicle cells is characterized by glycogen particles and microtubules. Figure 21a shows a connecting piece. It is closely associated with the basal plate and "boxes in" the proximal centriole. Portions of the connecting piece are also seen in contact with the distal centriole. The obliquity of section is responsible for the incomplete appearance of the connecting piece. Nine ribs approxi— mately 15 nm thick and 52 nm apart are visible. There are also nine ribs in the connecting piece of mammalian spermatids and spermatozoa. However in mammals it is.a much more complicated structure, with quite different dimensions (Winstatt g£_al., 1966; Blom and Birch- Anderson, 1965 and Fawcett, 1965). Figure 21b shows the relationship of the connecting piece to a centriole. The ribs appear to be extensions of part of a triplet. This very well may be the case since nine ribs can be counted in appropriate sections. 0“": - ._..-.L ‘- 76 Figure 21. Sections through connecting pieces, 3. clamitans. a) x 33,000. b) x 66,000. Note the connecting pieces (CP), basal plate (BP), mid-piece mitochondria (M), proximal (P) and distal (D) centrioles. In (b) only a single centriole (c) is present. 1. but «a.» 4 .0“! I .I q". n-I‘. O a ‘ I M,_ ‘IJ \ K [In 78 Extensions of the ribs are also visible in the interior of the centriole. Mitochondria are seen in association with the centriole for mid-piece formation. Various types of dense bodies are seen in the cytoplasm of spermatids during this phase of development. Figure 22 shows two types of such bodies. It is not known what these inclusion bodies are, where they come from or what they do, but they appear frequently. Type 1 dense bodies are membrane bound with less dense cores. They average about 85 nm in diameter and are not seen in any future stages. Type 2 inclusions are smaller, about 50 to 70 nm in diameter and often appear in packets or scattered in the cytoplasm. They are not membrane-bound and appear to be made up of finely granular material. More will be seen of these as develOpment proceeds. Note that the microtubules are arranged in a specific direction. A large amount of smooth endoplasmic reticulum is visible. With the increase in nuclear condensation the first indications of nuclear elongation are occurring (Figure 23). The microtubules are arranging themselves along the future anterior-posterior axis of the developing sperm. These microtubules may be partially responsible for the elonga- tion process. . By the time final nuclear condensation (but not nuclear compaction) has occurred, the first signs of acrosome formation are evident (Figure 24). The nucleus a "V 79 Figure 22. Inclusion bodies in spermatid, g. clamitans. Note the two types of cytoplasmic dense bodies (1 + 2), the mitochondria (M), microtubules (MT) and the smooth endoplasmic reticulum (SER) . x 26,100. Figure 23. Microtubules in elongating spermatid, B, clamitans. Note the microtubules (MT) and the centrioles (C) o x 17,400. 81 Figure 24. First signs of acrosome formation, 3. clamitans. a) x 15,300. b) x 42,000. Note the condensed but not yet compacted sper- matid nuclei (N), what may be the remnant of the nucleolus (Nu), preacrosomal vesicles (arrow), type 2 cytoplasmic dense bodies (2), inter- cellular bridge (IB), basal plate (BP), mito- chondria (M), axial filament (AF) and the Sertoli cell cytoplasm (Sc). 83 has a tightly packed dense granular appearance. The last remnants of the nucleolus are still evident. Large cisternal Golgi-like elements that may be rearrangement products of the smooth endoplasmic reticulum are seen (see Figure 25). No characteristic Golgi elements are seen at this stage and the origin of the Cisternae and the fate of the Golgi seen in previous stages (Figures 17 and 19) are not known. Many vesicles are now seen in the cytoplasm and the mitochondria are circular in section (Figure 24b). The cristae are much thinner than in the ndtochondria of previous stages and the matrix is denser (see Figure 21). Notice again the basal plate. Type 2 inclusion bodies are spread throughout the cytoplasm. Some, however, are in association with the centrioles. Dense preacrosomal vesicles are in close association with the anterior portion of the nucleus. The glycogen-rich Sertoli cell cytoplasm has separated the cells of the spermatid cluster. The intercellular bridges seem some- What disarranged. This is 'due to the separation of the cells by the invading Sertoli cell cytoplasm. Figure 25 shows the anterior portion of the nucleus prior to visible acrosome formation. The cisternal G"lgimlike structure is clearly visible. Note the differ- ence in size and density of the vesicles close to the a . . nterior portion of the nucleus. The veSicles appear 6 U quay n 7- :~: ‘ u‘ ‘ 1 Ole “up. ' '1 unit new. a . . N- 9..., '0‘ .0.- '.'.E .535: ”'7' Fun '6‘...) . I ..A‘ . ’ " "- bu F?“ ‘v... I'- :-,A:h" ..."V1 c ‘..b‘ ' ."'.o a a U 84 somewhat smaller and more dense the closer they are to thernucleus. Figure 26 shows the anterior portion of a sperma- tid which is actively engaged in acrosome formation. Note the large cisternal array and the membranous vesicles of various sizes and densities. Perhaps the vesicles arise from the cisternae in much the same way proacrosomal vesicles arise from the typical Golgi material in mammalian acrosome formation . At least three phases of acrosome formation can be distinguished. l) Forming phase: The cisternae give rise to the large clear vesicles. 2) Condensing_phase: The large vesicles then move toward the nucleus, condensing as they go. 3) Acrosome phase: When the vesicles finally reach the anteriOr portion of the nucleus they are ex- tremely dense and may now be called preacrosomal vesicles. It is apparent that all the vesicles in the cyto- zflesm are not involved in acrosome formation. The type 2 dense bodies seen scattered throughout the cytoplasm do “Qt aPPear to be associated with acrosome formation.« Figure 27 show progressive stages in the fusion of the Preacrosomal vesicles. It is not known exactly when Emeacrosomal vesicles can be called acrosomes but by the time a membrane bound sac covering the anterior portion of the nucleus similar to that seen in Figure 27c is seen, the ham - . ~ e immature acrosome may be appropriate. 85 Figure 25. Spermatid just prior to acrosome formation, 3. clamitans. Note the Golgi-like cisternal element (Cs) and the various type vesicles (V) around the nucleus (N). x 42,000. Figure 26. Forming acrosome, 5. clamitans. Note the cisternal element (Cs), preacrosomal vesicles (PA), and the type 2 cytoplasmic dense bodies (2). The three phases of acrosome formation; forming phase (Fp), condensing phase (Cp) and the acrosomal phase (Ap) are visible. x 42,000. 87 Figure 27. Fusion of preacrosomal vesicles, 3. clamitans. a) x 26,000. b) x 42,000. c) x 33,000. Note the fusing preacrosomal vesicles (Fa) at the tip of the spermatid nuclei (N), the cisternal element (Cs) and immature acrosome (IA). ‘2) :APQ. a A t..-'.'.‘ . K}- i 0“} “F noon '5 '.'.Z.'.S ' on ..R ‘ I Vigi. ~v.‘ A... . 1.. 5 RA u." by V... '. I. ”V- ‘I.: I h‘~.= ‘I 4'. s.‘ u A. a... :l.’ 8" (a 0" 89 Burgos and Fawcett (1956) described acrosome formation in the toad at the electron microscopic level as a fusion of vesicles. They state that the spermatids lack conspicuous Golgi elements, but believe that the Golgi is represented by one or more very small aggrega- tions of spherical vesicles of various sizes. No micro- graphs of this were shown. Sharma and Sekhri (1955) describe at the light fluoroscopic level Golgi elements in the spermatids of 53g3_ tigrina as.a few deeply staining granules scattered through— out the cytoplasm. These granules then coalesce near the centrioles to form what they call a proacrosome which migrates to a position at the anterior portion of the nucleus. This proacrosome increases in size to form the mature acrosome.” With acrosome formation, the nucleus continues to elongate (Figure 28). However, microtubules are no longer Present; in fact, most have been displaced to the posterior Portion of the cell in the residual cytoplasm which is now beginning to form. The invading Sertoli cell cytoplasm has co“‘Pletely separated the spermatids. The invasion of the cluster may also be responsible for the posterior movement of the spermatid cytoplasm. A small amount. of cytoplasm is etiJJ.visible at the anterior portion (acrosomal end) of‘uhe cell, very little being left in the lateral aspects andthe remaining portion in the posterior region. Most 90 of the posterior portion will become the residual cyto- plasm. Figure 29 shows a section through a portion of the residual cyt0plasm. It contains mitochondria, some of which seem to be deteriorating, microtubules, type 2 dense bodies, and Vesicles of various sizes and densities. Notice that the axial filament is in the central portion of the residual cytoplasm. With further development the residual cytoplasm becomes extremely dense and organelles lose their identity. Lipid droplets, mitochondria, ribosomes, endo- plasmic reticulum, Golgi, multivesicular bodies, multi- granular bodies and PAS-positive material have been described in the residual cytoplasm in a wide variety of mammalian species (Dauost and Clermont, 1955; Smith and Lacy, 1959; Lacy, 1960; Firlet and Davis, 1964 and Dietert, 1966). By the time the nucleus has compacted, that is, has 1°3t its granular appearance, the reSidual cytoplasm has reached the posterior portion of the nucleus (Figure 30). IntBreellularbridges and mitochondria are the only struc- tures still recognizable in (the dense cytoplasm. Portions of the maturing acrosomes are visible on the elongating nuclei”. Segments of the anterior excess cytOplasm are being eliminated into the Sertoli cell cytoplasm. The anterior excess cytoplasm may be analogous to the 91 Figure 28. Elongating spermatid, 3. clamitans. Note the immature acrosome (A), spermatid nucleus (N), residual cytoplasm (Rc) and the Sertoli cell cytoplasm (Sc). x 17,400. Figure 29. Residual cytoplasm, B. clamitans. Note the axial filament (AF), type 2 cytoplasmic dense bodies (2), vesicles (V), mitochondria (M) and microtubules (MT) in the residual cytOplasm (Rc). Portions of the Sertoli cell cytoplasm (Sc) and spermatid nuclei (N) can also be seen. x 17,400. 93 Figure 30. Nuclei in final stages of elongation, R. clamitans. Note the anterior excess cytoplasm (Ac), intranuclear inclusions (I), immature acrosomes (IA), mid-pieces (MP), centrioles (C), and the residual cyt0plasm (Rc) con- taining mitochondria (M) and intercellularw bridges (IB). Portions of the Sertoli cell cytoplasm (Sc) are also visible. x 10,200. 94 95 cytoplasmic droplet seen in mammalian spermiogenesis. In mammals the residual cytOplasm and the cytoplasmic droplet are not the same. In mammals, final maturation occurs in the epididymis with the elimination of the cytoplasmic droplet. (See Bloom and Nicander, [1961] for an ultra- structural analysis of the cytoplasmic droplet in mammalian species.) Frog spermatozoa reach final maturity in the testes (Rugh, 1951 and van Oordt, 1960). Thus, similar processes may be occurring at different locales in the different species. A large number of intranuclear inclusions are seen at this stage. For further discussion of this topic see the section entitled "Intranuclear Inclusions" p.182. With the final elongation of the nucleus the residual cytoplasm has proceeded over the mid—piece and has moved down the axial filament (Figure 31) . While this is occurring, the anterior end of the nucleus elongates into a finger-like projection upon which the acrosome finishes its development by fusion of the remaining acrosomal vesicles (Figure 32) . Figure 33 is a diagramatic representation of nuclear elongation and residual body formation. (a) represents an early spermatid just after axial filament fc"’-"‘3‘€lt:ion and just prior to nuclear elongation. The axial filer“ent‘extends through the sleeve-like process of the Spermatid cytoplasm. With the beginnings of nuclear 96 Figure 31. Mid-piece and tail with residual cytoplasm eliminated, R. clamitans. Note the mid-piece, (MP), axial filament (AF) in the tail, glycogen (G) particles associated with the tail and portions of the residual cytoplasm (Rc). Microtubules (MT) in the Sertoli cell (Sc) are also visible. x 10,200. Figure 32. Maturing acrosome, R. clamitans. Note the fingervlike projection of the nucleus (F), the large, still immature, acrosomal vesicles (IA), anterior excess cytoplasm (Ac) and intranuclear inclusions (I). x 33,000. 98 Figure 33. Diagramatic representation of the elimination of the residual cytoplasm. 99 100 elongation (b) the cytoplasm begins to collect in an area posterior to the nucleus. Note that the sleeve is no longer present. Line 1-2 is the proposed plane of section for Figure 29. Note that the axial filament in such a section would be repreSented in the middle of the cyto- gflasm. With continued nuclear elongation the residual cytoplasm begins to make its way past the mid-piece and down the axial filament. A longitudinal section through (c) may represent the section actually observed in Figure 30. Finally, in the fully elongated nucleus (d) the residual cytoplasm has made its way to the posterior end of the axial filament (see Figure 31) or has been eliminated into the lumen of the seminiferous tubule. Anterior excess cytoplasm has been eliminated in (d) of the diagramatic model. Regaud (1901), Lacy (1960), Firlit and Davis (1965) and Dietert (1966) have described in mammals the phago- Cytosis of the residual cytOplasm by the Sertoli cell and itsedegradation as it migrates toward the basement membrane. Dietert.(l966) and Br6kelmann (l963)‘have demonstrated acid Phosphatase activity in free granules and Golgi elements of theextruded reSidual cytoplasm. Dietert (1966) suggests that initial degradation occurs by lysosomal cytoplasmic autophagy. 101 No evidence of residual cytoplasmic phagocytosis by the Sertoli_cells in either of the two species of Rana studied has been noted. The final maturation product of spermiogenesis in R. clamitans can be seen in Figure 34. The nuclei are now long (15 to 17 um), slender and very dense. The acrosomes- have matured into bag-like structures at the anterior por— tion of the nucleus. The mid-pieces are formed and the residual cytoplasm has been eliminated. H Features Characteristic of Rana pigiens-Spermio-. genesis--The first noticeable difference in spermiogenesis between the two species concerns mid-piece formation. Figure 35 shows a portion of a spermatid cluster just after centriole migration and nuclear condensation. There is no indication of a connecting piece or basal plate as seen in R. clamitans spermatids of a similar stage of development (see Figure 21). The nuclear material condenses much earlier in R. pipiens spermatids. A similar stage of nuclear cOndensation is not reached in R. clamitans until the acrosomal formation stage (see Figure 24). The cytoplasm has many free and rosetted ribosomes. Microtubules are randomly oriented. The mitochondria are Oblong with parallel cristae. Those mitochondria not associated with the centrioles for mid-piece formation are located at the cell periphery. Few vesicles and no Golgi or Golgi-like elements are visible. Granular dense 102 Figure 34. Mature spermatozoa, R. clamitans. Note the acrosomes (A) on the elongated nuclei, the mid-pieces (MP) and tails (T). Sertoli' cell nucleus (Son) and cytOplasm (Sc), along with residual cytoplasm (Rc) and degenerating spermatids (Ds) are also visible. x 4,800. 103 104 Figure 35. Spermatid, R. pipiens. Note the condensed nucleus (N), proximal (P) and distal (D) centrioles, axial filament (AF), mitochondria (M), cytoplasmic inclusions (Ci), granular body (Gm) and microtubules (MT). Arrows indicate clumps of ribosomes. Glycogen (G) is visible in the Sertoli cell cytoplasm (Sc).' x 24,000. 105 106 bodies about the size of mitochondria and inclusions, somewhat denser than the surrounding cytoplasm, consisting of vesicles and granular material, are visible. The func- tion, origin and fate of these bodies is-not known. The Sertoli cell surrounding the spermatid cluster contains many a—type glycogen particles. There is a difference in density and size between the ribosome (18 nm) and the glycogen particles (28 nm). Figure 36a shews a cell in the same cluster as. that shown in Figure 35. The main purpose of this micro- graph is to emphasiZe the apparent lack of Golgi-like material._ The nuclear material shows signs of compacting and what may be a chromatoid body is present. Even with the beginning of elongation (Figure 36b) little or no change is observed in the cytoplasm. It may be noted that the spermatid cluster is retained, while in R. clamitans testes, clusters at similar stages are being broken up by the Sertoli cell cytoplasm (see Figure 24). However by the time the nucleus is partially compacted, the cytoplasm has changed considerably (Figure 37). B-type glycogen particles become visible. There is also an increase in smooth endoplasmic reticulum. Smooth endoplasmic reticulum has been described (Coimbran and Leblond, 1966; and Millonig and Porter, 1960) as structural elements associated with glycogen production. Glycogen has not been observed in R. clamitans spermatids 107 Figure 36. Spermatids, R. pipiens. a) x 17,400. b) x 15,300. Note the ribosomes (R), spermatid nuclei (N), chromatoid-like body (Cb) and axial filaments (AF). 109 Figure 37. Condensing nucleus, R. pipiens. Note intranuclear inclusions (I), smooth endoplasmic reticulum (SER), glycogen particles (G), mitochondria (M), vesicles (V) and a Golgi-like element (GL). x 26,500. 110 are». 4.9 a... “a. ... 111 at this stage. Mitochondria resemble those described at earlier stages (see Figure 35). Large clear vacuoles and dense membranes are also found in the cytOplasm. A Golgi- 1ike structure is visible. However, no association of this type of structure with acrosome formation could be made. L Figure 38 is a portion of a spermatid cluster at E a stage in development somewhat later than that shown in Figure 37. This is an oblique section through the elon- gating spermatids. The amount of glycogen, which has substantially increased, is being formed in pockets, often associated with membranous scrolls. These scrolls may be excess nuclear membranes discarded during the compaction and elongation processes of nuclear deve10pment (see Figure 39). Numerous vesicles and vacuoles of various Sizes and densities are visible in the cytoplasm. It is Possible that some of these may function in acrosomal formation. However, no evidence for this is available. The mitochondria are clustered around the centrioles for. mid-piece formation. Intracellular bridge, axial filament, microtubules and portions of the residual cytoplasm are all visible. Figure 39a shows the membranous scrolls with their doUbled membrane structure similar to nuclear membranes. Portions of the clustering glycogen particles are seen in association with these membranes. 112 Figure 38. Cluster of condensing nuclei, 3, pipiens. Note centrioles (C), residual cyt0plasm (Rc), intracellular bridge (18), axial filaments (AF), glycogen particles (G), membranous scrolls (S) and vesicles (V). x 12,500. 113 114 Figure 39. Membranous scrolls, R. pipiens. a) x 44,000. b) x 23,000. Note membranous scrolls (S), glycogen particles (G), axial filaments (AF), and microtubules (MT). 116 Figure 39b shows the orientated microtubules in the elongating spermatids. Membranous scrolls and clustering glycogen particles are also seen. With the elongation and posterior migration of the residual cytoplasm, the spermatids appear to emerge out of the cluster. The glycogen particles are arranged in packets of varying densities. Portions of the membranous scrolls are seen in association with these packets. Mitochondria, intercellular bridges, axial filaments, and vesicles are still visible in the dense residual cyto- plasm (Figure 40). The residual cyt0plasm with the massive amounts of glycogen is eliminated down the tail (Figure 41). In Uanother portion of the cluster a partially elongated spermatid is seen with the residual cytoplasm at the area just posterior to the nucleus. Notice the size and density nmmno macs macomoumfinmmm mumEHHm N 0mm» masomoumaummm mHmeum m 00%» macomoumEnmmm mumsflum N mama mfisomoumeummm aumEanm N 0mm» owummo mHHmo “masomucmumsm an conesOHHSm Um>ummno mcoc macomoumeuwmm mumeflum N mean mwsomoumEMwmm humaflum N wax» macomoumeuomm aumaflum m mam» macomoumEummm humswum m 0mm» Oflumho mcoauMfiUOmmm Hamo Hmwcomoumshmmm mmwpon vacaamummuo UHEmchouao Hmaosc Oflnmuoaaaom Ummoam>m© ummn =ucmfimo= Hmflncsonoouwenmucfl coaumohaahuass Huang Iconoouflfi.mo woman oases» cw mHHmo Hmcflfinmm unwound Am Am Av Am Am AH mfimwcumwumooumfiuumw mammcmmoumsummw mm GMNB mHmEEmz mcmwmam am mcmuHEmao .m mAnmmno 0:0: .om>uomno econ mmaowmm> xom AH mmmcmoum oauoflmEmum mamsamz msmfimwm am mcmuwewao am 220 acmmmum m + m + m haco Hmfifixoum asmmmnm macaa Imaum> mmaommm xaamn mcaEHOM .mmaamcmmuo pmmsm acmmnm =umammcon= Umcmaamam acmmmum msmaosc mamaum>o =mmn= msomchOEoc asmmnm m + m Hmamap + Hmfiaxoum asmmam moumom mmHHmcmmuo Hmsoa>apca asmmmum =m>aaafiaam= Hmoauocaaao asmmnm msmHosc mo maa oa vmcamcoo Ammaoamm> no mxomam .mHscmumv pmcwfiumamp aoc acmmnm m + m HmamHU + Hmfiflxonm acmmmum Hmnao 30mm oa Hmaamuumm .mcmE mmHHmcmmuo Hmspa>aoca asmmmum =m>waafiaum= Hmoanocaamo acmmmum msmHosc mamHHm>o =mmn= msomsmmOEos madness Hama mmaoanacmo momwm mcaaomccoo mmamauo Hmaucconooaafi wauoconooaae momamlpaa cw ammoo>am mama momamloae msmaosc mo mmmsm mommm HmEOmOHOMQSm mEOmouom mo coaaamom GEOMOHUM ANH Add Aoa Am Am An Am Am Aw Am AN mo musaosnam AH moNoamEhmmm macaw: mHmEEmz mcmwmdm am mamawfimHo .m (“i 1) 2) 3) 4) 5) SUMMARY The ultrastructural aspects of spermatogenesis were studied in two species of 3333. Spermatocytogenesis, that portion of the meiotic cycle studied here and the early phases of spermiogenesis are similar in the two species. These processes were com- pared to the similar stages described in mammals. Acrosomal formation and the final structure of the acrosomes and mid-pieces are different in the two species. These variations were compared with similar published accounts of amphibian material. Sustentacular cell development was studied. Histochemical localization of glycogen and acid and alkaline phosphatases was carried out. 221 LITERATURE CITED LITERATURE CITED Adams, E. C. and Hertig, A. T. (1964). Studies on guinea pig oocytes. I. Electron microscopic observations on the development of cytoplasmic organelles in oocytes of primordial and primary follicles. J. Cell Biol. 33, 396-427. Allison, A. (1967). Lysosomes and disease. Sci. Amer. 21?, 62-730 Anberg, a. (1957). The ultrastructure of the human sper- matozoon. Acta Obst. Gynecol. Scand. 33. Suppl. 2, 1.133. 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