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The seminiferous epithelial cycle and spermat ogenesis in rams (ovis aries )
THERIOGENOLOGY
THE SEMINIFEROUS EPITHELIAL CYCLB AND SPERMATOGENESIS INRAMS (CVIS ARIES > --
G.S.Bilaspuri and S.S.Guraya Department of Zoology,College of Basic Sciences and Humanities, Pun ab Agricultural UniverSity, Lud!! iana-141C04,Pun5ab,India Receivedforpublication: August 27, 1984 Accepted:February 28, 1986
ABSTRACT The efficiency of spermatogenesisand degenerations of different spartuatogenicCells Under nOIIUa1conditions of ths environment have been Investigated In rams. The melotic divisions and the position of first-generation spermatids in haenatoxylin-eosin stained testicular preparations were used to ldentif eight stages of the Seminiferous epithelial cycle (SBC3 . The stages of relatively long duration (i.e., 1,2,3,4,8) were sub-divided. The percentages of frequency for the 14 stages reported were also studied. Three generations of type A (Al, ,A3’90= generation of type intermediate (In) and2 wo generations of type B (B ,B > spermatogonla were recognized. A2 and B spermatog&ni$ as well as primary and secondary s$ermatocytes did not degenerate. Contrarily, A A3, In and B spermatogonia showed 25 13.7 27.3 ahi % 2 124% degenerations respectively. We colluded that compared with the previously used eight-stage classification,subdivi.dingstages wZth long duration as done In this study facilitates investigating the degenerations of sperrnatogcnfc cells. The efficiency of spermatogenesis In rams was 47.58$since one A spermatogonium produces 30.45 spernatids/spermatozoa aJainst the expected number of 64. Key words :
spermatogenesis, quantitative spermatogenesis, efficient of spermatogenesis, degeneration of sperma9: ogenlc cells, rams
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THERIOGENOLOGY INTHCkDU~TION Spermatogenesis is a continuous process taking place in various cellular stages which follow each other in time. Each stage consists of one or two generations of spermatogonia, spermatoc tes, and s rmatlds in addition to Sertoli cells. OnPy a limltegenumber of spermatogcnic stages and sub-stages could be seen in a cross section of a tubule. Based on meiotic divisions and the position of firstgeneration(more mature) spermatids, 8 stages of SEC have been identified In several mammals including rams(l-10). The relatively long-duration SEC stages have been subdivided in bull (11) and buffalo (12); identification of this sub-division has increased the specificity of this classification method, and consequently, the quantitative spermatogenesis (13) and cytochemical changes in spermategenie cells could be studied more precisely (14). In contrast to this classification method, the developent of acrosome and spermatid nucleus during spermatogenesis as seen in periodic-acid Schiff-haematoxylinstained preparations has been employed to study the efficiency of spermatogenesis in bulls (15) and gpats (16). With the processes described above,the present study was undertaken to sub-divide the already known eight stages of SEC identified in haematoxylin-eoslnpreparations in rams. The efficiency of spermatogenesiswas worked out by counting the different spermatogenic cells In %hese stages/ sub-stages. MATERIALS AND METHODS The left testis was obtained from 17 sexually meture (20 yr) rams (Ovis aries) The tunlca albuginea was removea and placedYiiXo-Z-7X9$saline solution; two or three small pieces (A-l.5 cm3) from each testis were fixed for 24 h in freshly prepared Zenker's fixative containing 10% formalin. The tissue was washed in running ta water for 24 h dehydrated treated for removal of ioil ine,a%ziembedded in paraffih wax (M.P. 58-60%). The sections were cut at 5 p and stained with baematoxylineosin (17).
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The meioticdiviiions,morphology and position of spermatids of the first generation were used to define eight main sta es of SEC. %ed. In the subStages l,2,3,4 and 8 were sub-divi stages of a given stage, the spermatogonia were of the s.ametype; however, other cell generations varied to differ%@g degrees. The relative occurrence of various stages and sub-stages was investigated by identifying the stage of the cycle in 2850 round tubular cross sections selected at random from different animals with at least 100 tubules per animal. From this, the rcentage of frequency was calculated.For the quantifs" ative behaviour of various spermatogenic cells, whole nuclei as well as their identifiable fragments were counted at each stage in at least 80 round seminiferous tubules. The dimensions of each cell type were recorded with the help of stage micrometer and occular micrometer. The counts of various germ cells were corrected for nuclear diameter, which may vary with thickness of a sectian. The correction was done by Abercrombie's formula (18) as outlined by Clermont and Morgentaler (13). For each cell type, the arfthmetic mean and standard error were calculated in each stage. The small fragments of Sertoli cell nuclei were not counted. Therefore, their number was not adjusted for nuclear diameter. FlESlJLTS
The salient features of various stages and substages are described below. The morphology of various spermatogenic cells was basicall, similar to that described already for the ram (25 . Eut in the present study based on the nuclear size and shape, zy o;t.;; and diplotene could be divided into their earlya n% phases; leptotene consisted of leptotene roper and granular lcptotene, which were similar to Phose in bull (11) and buffalo (20). Additionally, three generations of type A spermatogonia were distinguished in our study, in contrast to only two such gener t+z;zo;;ported by Ortavant (2). The nuclear dimensions oa spermatogenic cells are given (Tables 1 and 2). The descri tion for the morphology of cells is given briefly only wRere it is required.The distribution of various cell types and the comnosition of various stages/sub-stages are likewise summarized (Table 3).
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Table
1.
Variations in spermatogonial clear diameter ( p 1 and volume ( p ? in
ram Spermatogonia
Stages
Diameter
Volume
1-8
8.05
273
3b-4 2 g -la lb
7.40 8.23 8.00 8.50
% 291
z;.
3x 406
2a 2b
7908 7.44
186 216 136
223 Xn
4
6.80
165
B1
7
6.48
142
B2
8a-la
6.13
120
Spermatogenic
Stages
Stage 1. From the liberation of spermatozoa into the tubular lumen until the appearance of leptotene s ma tocytes (Plate 1, F’igures 1-3). This stage was c r aracterized by the presence of only round spermatlds near the tubular lumen. Stage la. From the liberation of spermatozoa into the tubular lumen until the end of type B2 spermatogonia (Plate 1, Figure 1). Stage lb. From the end of mitosis of type B2 rmatogonia until the end of the preleptotene f IF age (Plate 1, Figure 2). until
488
Stage lc .F’rom the end of the peleptotene the appearance of leptotene (Plate 1 ,Mgure
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Table 2. Variations in germ cell nuclear diameter (p) and volume ($1 in rams
ceutype
Diameter
Volume
Spermatocytes Preleptotene Leptotene Granular leptotene Zygotene 1 Zygot3ene 2 Pachytene 1 Pat hytene 2 Diplot ene 1
5.69
6.13 6.39 6.83 ;:!4
8.31
89%
Secondary Diplotene spermatocyt&. 2 9.652
97
127
137 1
!% :I6 303
iii'
14z
S permatids 1. Spermatids (Stages 4,s) 2. Spermatids (Stages 6,8)
5.08
69
50%
90
Diplotene 2/ A, Spermatogonium (dtage lb)
1 *OS
1 loo
Diplotene 2/ 1 Spermatid
:*$?I .
MI
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THERIOGENOLOGY Table 3. The percentage frequency of stages of SEe and their cellular composition in the rams Stage la lb Ic
3 frequency
Sperma-, b permatocytes% rmids togonia 1 oung UQ Y%$-C
III
6 45 1 1;:;: . j 22.23
!"5;:j 10.78 .
A2 A2
GL z1
3a 3b
'6'*5;] 17.60 .
A3
=2 Z2
4a 4b
';*G?',10.61
In
z2
A3 IV
l
4.35
5
D, D,
D;!‘D, Ed SP
4.35
Ifi
c1
6
12.67 12.67
In
*?
7
11.03 11.03
B1
*1
El El
II R
Ed
R
Ed
R
Ed
R Ed B2V pl R Ed PI B2 A snermatoaonia were also present in all stages 1. eficeptin SEages 2a and 2b.II. PL, preleptotene; L,proper leptotene; GL,granular leptotene; Z,,early zygotene; Z2,1ate zygotene;P ,early pachytene; P ,late pachytene;D early diplotene;$2, late diplote%e; DI,diakinesis;ah-rI,secondary spermatocytes. III R round spermatids;El,elongatingspermatids;Ed, eiongaoed spermatids. IY. Some Xn spermatogonia also started appearing in stage
65*?5{ 11.73 .
2 4r
”
v.
490
$&matogonia B2 very rarely divided even ih Stage 8b to form preleptoLene spermatocytes.
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titage 2. Prom the appearance of granular in grains,’ until the leptotene, i .e . lleptotene appearance of zygotene (Plate 1, Figures 4,5). The chromosomes of primary sperma W* ocy es at the leptotene stage were leading to the formation of ‘leptotene in grains’ due to the appearance of numerous granules in them. The older primary spermatocytes, which were previously a t the pachytene stage , were now in the early diplotene stage. The nuclei of spermatids began to elongate (Plate 1, Figure 4). Sta e 2b. Some primary spermatocytes had e early zygotene whereas older spermatocytes enteree were at the early diplotene stage. The nuclei of elongating spermatids were oriented towards the basal membrane of the seminiferous tubule (Plate 1, Figure 5). Some primary spermatocytes, which were previousV+ y a the late leptotene or early zygotene stage, had now advanced to the late zygotene stage.Tbe older primary spermatocytes and spermatids continued to increase in size (Plate 1, Figures 6,7,15). All the primary s permatocytes previously at the v*ear y zygotene stage advanced to the late zygotene stage. The older primary spermatocytes remained at the early diplotene stage. The nuclei of elongating spsrmatdds grouped in bundles and became embedded in Sertoli cell cytoplasm (Plate 1, Figure 6). Sta e b. The older spermatocytes increased in size t+ ecome the late dlplotene spermatocytes. The elongated spermatids attained their maximum length( Plate 1 ,Figure 7). Stape 4. Eegan with late diplotene/diakinesis spermatocytes and proceeded until the end of both meiotic divisions (Plate 1, Figures 8,s).
the
Stage 4a. The primary spermatocytes after a very short diaklnesis or late diplotene stage had already divided. The present stage ended \!ith the disappearance of prLmary spermatocytes that were earlier at the diplotene or diakinesis stage (Plate 1, Figure 8 > .
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Stage 4b. This stage was characterized by the absence of primary spermatocytes at the pachytene, diplotene,and diakinesls s ta es. Consequently, the primary spermatocytes of the $i rst, or older, were absent .The secondary spermatocytes generation and the figures of the second maturation divisions were present (Plate 1, Figure 9). The nuclei of young spermatids were incomp etely developed and possessed the grains v* of chromatin connected together by a fine thread.The younger spermatocytes, whichwere in the late zygotene stage, progressed i&o the early pachytene stage (Plate 2, Figure 10). Stage 6. The nuclei of young spermatids increased in size. Their chromatin of fine granules contained 3 or 4 karyosomes (Plate 2, Figure 11) . The bundles of elongated spermatids migratW* owards the lumen of the seminiferous tubules(Plate 2, Figure 12). Stage 8. F’rom thr; complete bordering of the seminiferous tubule with the elongated spermatids (spermatozoa)until the liberation of s ermatozoa into the lumen of the seminiferous tubule ( %1ate 2,F’igures 13,141.
The elongated spermatids eompletely border%%??* umen of the seminiferous tubule as Immature spermatozoa (Plate 2, Figure 13). elongated spermatlds were progressthe lumen of the seminiferous spermatozoa (Plate 2,Figure 14). Percentage tiequency of Stages of SE0 In the first four stages (containing one generation of spermatida percentage frequency was 61 .12$ compared to 3 in the last four stages(contalning two generations of spermatids)(Table 3) .This finding was similar to that in rams(21), bulls (11) and buffalo (121,but dlsslmllar to that of rats(l). Spermatogenlc Cell. Counts The dimensions of the nucleus in different germ cells are glventtables 1 and 2).The average number of SperaratogenM and Sertoll cell nuclei at various stages of SE and the number of tubules enamined for each stage follow(Table 4). 492
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OLl OZl
W1
OCL
pa
:o9t
06
g*zTg*sz
L’L+Q’ZL
9’ lZZ.OZ z*l~Z’oz
$:!%:I
OL 91 -“1
92 =f 92 w
$
8:oz8:8
98
=8 L 9
THERIOGENOLOGY EXPUNATIONOF FIGURES A part of seminiferous tubules show the cellular associations in different stages of the seminiferous epithellal cycle In haematoxylin-eosin stained sections from a portion of testes of rams (X 1080; Figures ,l-14; X 2160, Figure @>.The lumen of each tubule is on right hand side. PLATEIa Figures
Stages la, lb, and lc of cycle differ from one another in having B spermatogonla,prelepto&ene a&
l-3.
spermatocytes respectively towards periphery. The most characteris tls
the
feature of these stages is the presence of only one generation of round sperm+ tids. Figures 4-T.
Stages 2a and 2b which differ in showing granular leptoteneand early zygotene phases of second generation of spermatocytes; the first generation of spermatocytes has advanced to early dlplotene.The elongation of spermatlds starts in stage 2a and continues in stage 2b.
Figures 6-7
l
Stages 3a and 3b differ in the presence spermatids and the elongaof elongatin ted spermatl %s which attained maximum length .The first generation of spermatocytes from early diplotene In stage 3a advances to late dlplotene In stage
3b.
Figure 8.
Stage 4a shows metaphase,anaphase telophases of meiosis-I.
Figure 9.
Stage 441characterized
aLnd
by the presence of secondary apermatocytes ,meiosis-II and young spermatlds and by the absence of primary spermtocytes of first
generation.
lanation aExp
494
of abbreviations follows Plate 2.
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Plate 1
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THERIOGENOLOGY
Figure 10.
Stage 5 showing primary spermatocytes advanced to early pachytene,the young round spermatids with incompletely developed nuclei are preseti throughout the seminferous tubule in addition to first generation of elongated spermatlds
Figure 11.
Stage 6 showing nuclei of round spermatids with increased size;the other cell types being similar to those in stage 5.
Figure 12.
Stage 7 characterized by presence of B, spermatogonia and the movement of bundles of elongated spermatids towards the tubular lumen.
Figure 13.
8a characterized by the bordering of tubular lumen by immature SpermatoWa. B spermatogonia are present.Residual bgdies are also seen. Stage 8b characterized by the presence of immature spermatozoa at isolated pla.ces. The other cell types are similar to those of stage 8a.
Figure 14.
Figure 15.
Stage
k spermatogonia In association with late diplotene and late zygotene, elongated spermatids of stage 3b.
aFxplanation of abbreviations follows Plate 2.
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Plate 2
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Abbreviations in plates different types of A spermatogonia different types of B spermatogonia early diplotene late di_plotene elongated spermatids EL
elongating speWids
GL
granular leptotene
In
intermediate spermatogonia
L
leptotene
W-I
figures of meiosis-I
M-II
figures of meiosis-II
PI
preleptotene
p1 P, L
early pachytene late pachytene
EB
residual bodies
RS
round spermatids
S
Sertoli cells
ss
secondary spermatocytes
SZ
spermatozoa
YS
young round spermatids
z1
early zygotene
z2
498
late zygotene
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A type spermatogonia bad slightly elongated nucleus conta1ning fine dust-like cbromatin and were cbaracterlzed by the presence of only one nucleolus. These cells were missing only in stages 2a and 2b of the cycle. An average of 2.0 type AI spermatogonia were present in Stage lb which became 3.9 (Stage 2a) and 7.1 (Stage 3a), showing approximately twice the increase in their number in each of these stages. This indicated the occurrence of two mitotic divisions of t e A spermatogonia during these stages. The count of ce9Ps suggested that the major part of these mitotic divisions occurred between Stages lb-lc and 2b-3a. The combination of morphological observations with the counting studies suggested that all Al spermatogonia divided to form A2 s ermatogonia which subsequently divided to form a majcrley of A spermatogonia (Plate 2, Figure 15) and some Al spermatogon?a. 4 spermatogonia could be distinguished from the parent A spermatogonla because they were smaller in size snd they usually contained more than one nucleolus. Compared to A spermatogonia showed a relatively dark2 spermatogonia of stage 3a were onla but in stage 3b they increased in size (Table 1). A, spermatogonia showed a progressive increase in their size from Stage 3 until the end of the cycle(Table 1); boweoer,tbeir number decreased from 2.0 to 1.5 (Table 4), demonstrating a 25% degeneration of A type spermatogonla during these stages. In the second half of the cycle, the nuclei of these cells fragmented, and the celkultima~ely degenerated. Subtraction of A type spermatogonia in Stage 4a(A > from those of Stage ja (A +A > iridicatedthat 5.1 of Ai spermato:onia produced 8.2 IJIspermatogonia in Stage 4b. The assumption is made that the number of A spermatogonia that did not divide to form In spermatogonia remained constant in Stages 3 and 4. The total number of A type spermatogonia in Stage 3b was not considered because some In spermatogonia also appeared in this stage. Furthermore, In spermatogonia contained more darkly stained nuclei with coarse chromatin in their centers and on their nuclear envelopes than A spermatogonia. The nuclei of In spermatogonia te& ed to be smaller, more spherical& cohtained more than one small nucleolus. Although the number of In spermatogonia was maximal in Stage 4b , this sum was
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less than twice the number of the parent 1:type cells (Table 4). Thus, a 13.7$ degeneration of A spermatogonia was shown before the In spermatogonia 3ere formed. The number of In spermatogonia remained almost constant throughout their existence until Stage 6 suggesting the existence of only one generation of these spermatogonia in the SEC. In Stage 6, 8.8 In spermatogonia formed 12.8 El spermatogonia, the number of which was also less than twice the number of the&r parent cells. This indicated a 2'7,3$ degeneration of In spermatogonia, In the beginning of Stage 8a,the Bl spermatogonia divided to produce 20.2 spermatogonia.This number was again less than twice the number of parent cells, suggesting a degeneration rate of 21.2%. The division of RI type sT:ermatogoniato form a second type of F spermatogonia was coupled with morphological observations that showed the existence of two generations of 1?spermatogonia, Bland B . The nucleus of B in contrast to In type spermat5 gonia, tended to bg'smaller, more spherical and more darkly stained; it also contained a greater number of nucleoli which were smaller.The more darkly stained nucleus was due to the increased amount of chromatin crusts. B spermatogonia generally divided at the end of Stage la, o!$ very rarely at Stage 8b, to form twice the number of primary spermatocytes that showed a constant number throughout the various @ases of meiosis at all the stages of SEC. This suggested that the second generation of B spermatogonia and primary spermatocytes do not degenerabe. In Stage 4a, 78.5 secondary spermatocytes were produced. This number was almost twice the number of primary spermatocytes indicatin no degeneration of spermatccytes had occurred during firsI meiosis. To count the number of secondary s~r-~tocytes _t~~bn?eswith only secondary spermatocytestrare situation) or secondary spermatocytes with dlplotene or diakinesis (Stage 4a) were considered.After adjusting for the number of nuclei of these primary spermatocytes, their number was doubled to get the number of secondary spermatocytes; it was assumed that spermatocytes did not degenerate during these divisions. From 78.5 secondary spermatocytes in Stage 4b, l!?% Step 1 spermatids were produced. This showed that there was no degeneration in meiosis-II. The number of round sperm&ids during SEC apparent1 was not constant* the small increase or decrease in tK eir number did not follow
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any regular trend and suggested that these small variations in their number could be due to counting errors and not to degeneration. The number of Sertoli cells was constant throughout SEC (Table b),suggesting that there was no division in adult testis. Throughout the various stages, their number could not be adjusted for differences in nuclear diameter because of the changing shape of the nucleus within the varloua stages. The nucleus lay along the basement membrane forming a triangle with it in Stages 8b to 2b(PLate 2, Figure 140 Plate 1,Figures I-5). During the detachment of elongated sperrratidsfrom the Sertoli cells in Stages 6 and 7 (Plate 2, Figures 11,12), some of the nuclei took on a spindle-shaped appearance while most of them were flattened against the basement membrane. DISCUSSION Eight sta es of SEC have also been reported in some mammals inclu%ing rams (see references in the Introduction). But the subdivision of various stages with relatively long duratlon(Stages 1 ,2,3,4,8) have been previously done only in bull (11) and buffalo (12). If each sub-stage in rams were raised to the level of a stage, the total number Eventien subof stages in a cycle would be 14. divisions were based on the development of acrosomes requiring additional stages, only 12 stages have been reported in bull (15); however, using the outlined procedure, we have reported 14 stages in buffalo (22) and goat(l6). AS in our present study on rams, the Sertoli cells in other species have also been reported to undergo durin SEC and these cells have variations in nuclear sha also not divided in the aze ults (If ,15,16,23).
Due to the occurrence of six peaks of spermatogonial mitosis in SEC stages, we were able to identify six generations of spermatogonia. Out of these,three generations A ) one of In spermatogonia, were of A spermatogonia (A and two of B spermatogonia'1BFh2)? &eviously , only two generations of A sperma_togonia~A1,&?) and a total of only five generations were reported in rams (13 and in bulls (3). In contrast, there were six spermatogonial generations in bull (ll), buffalo (l3,2O), and goat (lG).The number of generations of various spermatogonia varies with the species studied and with the investigator(2k).
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The cell-specific degeneration observed in this study has also been re orted for certain cell types in some mammals (23,2$), but the systematic study of the behaviour of all spermatogenic cells has been done only in bull (I51, buffalo (13),md goat (16). The present observations have shown 25, 13.7, 27.3 and 21.25
degeneration
of types
A
A
In and R
spermatogonia, respectikely, 1; r&s.Calcula%ed from the data of Eerndtson &:Desjardins (15),these cells show 33, 6.3, 27.8 and 16.2$ degeneration rate in bull. In buffalo, these cells show 20, 18.75, 10.2 ard 15.5% degenera ion, respectively (13) compared with 26.3, 15, 25, 25.8k degeneration of corresponding cells in goat (16). As in the cited studies no degeneration could be observed throughout the meiotic popbase in mice (25), rats (26,27), bulls (3,15,28) and buffalo(l3). In our study, no degeneration was observed during the division of secondary spermatocytes to form spermatids. In contrast, there was a 18.7, IO and 10.4% de eneration of secondar spermatocytes respectively in bullf15), buffalo (133;, and goat (16). The deleterious effects of long daylight photoperiod on some spermatogenic a cells in rams have been studied (29). This study reported 19 and 23$~degeneration during the divisions of A and In spermatogonia, in addition to 36 and 4% degenkations, during earl pachytene and reduction ~~:Pz%l us the extene of degernration in rams differs according to normaland long daylight conditions. Due to their elongation and uneven distributioqthe number of spermatids could not be counted after Stage lc,Because of chromatin condensation and other some spermatids may degenerate ;~~;~e';"~~g$;g~~ 8 as has been reported for other species (26). The extent of degeneration that occurs at various steps of spermatogenesis becomes clear by the mean number of spermatids produced per A spermatogonium available for division following Stage 3a . This was only k7.58$ of the number expected inthe absence of degeneration. This number has been re ortdd to be 43 43.32,znd 33.1s in bull (15) buffalo ?13), and goat (16) respectively. If the possibility of degeneration during spermiogenesis was not considered, one A spermatogonium available for division after Stage 3b praduced 30.45 spermatozoa in rams instead of the expected number of 64. One A3 spermatogonium has been reporter?to produce 27.73,
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28 and 21.1 spermatozoa in buffalo (131, bull(l'j)and
goat(16) respectively. A spermatozoal loss has been reported in bulls during trans rt through the . It is concluded, epidldymis and vas deferens (30p" therefore that based on an ei ht-stage classification, the sub-division of sta es wi!h relatively long nvestigating the differentiaduration is useful for f: tion of spermatogenic cells. Our study has also found that spernatogeaesis in the ram is more efficient than that In the bu11(15), the tiffalo(l3) and the goat(16).
1. Roosen-Runge, E.C. and Giese1,L.O. Quantitative studies on spermatogenesis in the albino rat.American Journal of Anatomy & : l-30 (1950). 2.
Ortavant,R.Le cycle spermatog&&ique chez le b&er. D.Sc. Thesis. University of Paris,Paris(l958).
3. Amann, R.P. Reproduction capacity of dairy bulls.IV. Spermato enesis and testicular germ cell degenerafion. American Journal of Anatomy 110: 69-78(1962). 4. Hochereau,M.T. Constance des frequences relative des stades du cycle de 1' epithcyliumseminifere chez le taureau et chez le rat.Annales de Biolo e animale,Biochimie,Biopbysique 2: 93-102 (1963E
5. Swierstra, E.E. and Foote, R.H. Cytology and kinetics of spermatogenesis In the rabbItJournal of Reproduction and Fertility & 309-322(1963). 6. Swierstra,E.E. Cytology and duration of the cycle of the seminiferous epithelium of the boar;duration of spermatozoon transit through the epididymis. Anatomical Record m:171-186 (1968). 7ASwlers tra, E.E., Gebauer M.R. and Pickett,B.W. Reproductive physloiogy of the stalllon.I.dpermatogenesls and testis composition.Journalof Reproduction and Fertility 9:113-123 (1974) 8. Tiba,T. Ishikawa,T. and Murakomt,A.Hlstologiche u n&ersuchung der kinetik der spermatogenese beim Mink (Mutele vision .I&emenepithelzyklus in der Paarungsze 1G;J1apanese Journal of Veterinary Research 15:73-87 (1968).
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9.
Foote,R.H.,Swierstra,E.E.andRunt,W.L.Spermatogenesis in the dog.AnatomicalRecord u:341-352(19729.
?O.Schuler, H.M.and Gier,M.T. Duration of the cycle of seminiferous epithelium in the prairie vole (Microtus ochronaster ocbroaaster).Journalof Experimental Zoology,?q2:1-12(?976). ?l.Bochereau-de-Reviers,N.T.Etudes des divisions spermatogoniales:etdu renouvellement de la s ermatogonie souche chez le taureau.D.Sc. tResis,University of Parls,Paris (1970). l2.Guraya S.S.& Bilaspuri,G.S. Sta es of seminiferous uration of epithelial cycle and relative 6: spermatogenic processes in the buffalo.Genenbaurs or hologisches Jaharabuch Leipzig 122:147-161 19 6). ?? 13,Bilaspuri,G.S.and Guraya,S.ti.Quantitative studies on spermatogenesis in buffalo (Bubalus bubalis). Reproduction,Rutrition,De'veloppementm-982 (1980). 14.Bilaspuri,G.S.Morpbologicalhistochemicaland biocbemical studies on the testes of some farm animals. Ph.D. thesis Punjab Agricultural University, Ludhiam,Ind$a (1978). l!j.B@~?ndtson W.E. and Desjardins,C. The cycle of seminiferous epitbelium and spermatogenesis in the bovine testis.AmericanJournal of Anatomy. 140 :167-180 (1974). 16.Bilaspuri,G.S.and Guraya,S.S. The seminiferous epithelial cycle and spermatogenesis in goats (C r hircus).Journal of Agricultural Sciences, CZ%!d~RJ:368( 1984). 17.Humason,G.L. Animal Tissue Techniques.W.H.Freeman and Company, San Francisco (1967). 18.Abercrombie,M.Estimation of nuclear po lation from sections.AnatcmicalRecord&r 238~2@(1946). .Clermont,Y.and #orgentaler,H. Quantitative studies of spermatogenesis in the hypophysectomlzedrat. Endocrinology B:369-382( 1955). Spermatogenic cells and Bilaspuri,G.S. 20. Guraya S.S. of {be buffalo(Bubalus bub lis)testls.Indin Journal of Anlm~nces +368-375(1976!. _ 19
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APRIL 1986VOL.25NO. 4
THERIOGENOLOGY 21
.Ortavant,R.Spermatogeneslsand morpholo y of the spermatozoon.In:Cole,H.H.and Cupps,pi.T.(eds). Reproduction m Domestic Animals.Academlc Press,New York,l959,pp.l-50.
22.Guraya,S.S.and Bilaspuri G.S.Stages of seminiferous epithellal cycle in the buffalo.Annales de Biolo le animale,E%ochimle,Blophysi~ue 16: ,3791$4 (1976). 23.Courot M., Hochereau-de-Reviers,M.T.and Ortavant R.Bpermatogenesis. ns Johnson,A.D. Gomes,W.R. and Van Demark N.L. 5 eds.).The Tes&,vol.I, Academic Press,New York, 1970.~~ 339-432. 24.Clermont,Y. Kinetics of spermatogenesis in mammals: Semidf&~us epithellum cycle and spermato onlal renewal. Physiological Reviews &:198-236?1972). 25.0akberg,E.F.Sensitivltyand time of de eneration of spermatogenetic cells Irradiated in various stages of maturation In tht:mouse.. Radiation Research _2:369-391( 1955). 26.Clermont Y.Cuantltative anelysls of s rmatogene&s of the rat: A revlaed model for tI? e renewal of s ermato on1 .American Journal of Anatomy mt l&-,29( f962?. 2c/.Desclln,J,andOrtavant,R. Influence des hormones gonadotropes sur la duree des processus spermatog&&iques chez le rat.Annales de Blolo le anlmale, Biochimle,Blophysique2: 339-&k (1963). 28.Attal,J. and Courot,M. De'veloLxnent testlcularie et Establishment de la spermatogenese cheze le taureau.Annales de Biolo le animale,Biochemie, Biophyslque 3~219-241 (1663). 29.0rtavant,R. Action de 13 dure'edleclairemen;tsur les processus spermatogenetiqueschez le belier. Competes Rendus des Seances de la Soclete' de Biologie et de Ses Finaliales =:471(19563. 30.Amann,R.P. and Almquist J.O. Reproduction capacity of dairy bulls.VI.Ehfect of unilateral vasectomy and ejaculation frequency of sperm reserves; aspects of epididymal p4ysiology .Journal of Reproduction and Fertility 1:260-268(1962).
AFWL1986VOL.25NO.4
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THE SEMINIFEROUS EPITHELIAL CYCLB AND SPERMATOGENESIS INRAMS (CVIS ARIES > --
G.S.Bilaspuri and S.S.Guraya Department of Zoology,College of Basic Sciences and Humanities, Pun ab Agricultural UniverSity, Lud!! iana-141C04,Pun5ab,India Receivedforpublication: August 27, 1984 Accepted:February 28, 1986
ABSTRACT The efficiency of spermatogenesisand degenerations of different spartuatogenicCells Under nOIIUa1conditions of ths environment have been Investigated In rams. The melotic divisions and the position of first-generation spermatids in haenatoxylin-eosin stained testicular preparations were used to ldentif eight stages of the Seminiferous epithelial cycle (SBC3 . The stages of relatively long duration (i.e., 1,2,3,4,8) were sub-divided. The percentages of frequency for the 14 stages reported were also studied. Three generations of type A (Al, ,A3’90= generation of type intermediate (In) and2 wo generations of type B (B ,B > spermatogonla were recognized. A2 and B spermatog&ni$ as well as primary and secondary s$ermatocytes did not degenerate. Contrarily, A A3, In and B spermatogonia showed 25 13.7 27.3 ahi % 2 124% degenerations respectively. We colluded that compared with the previously used eight-stage classification,subdivi.dingstages wZth long duration as done In this study facilitates investigating the degenerations of sperrnatogcnfc cells. The efficiency of spermatogenesis In rams was 47.58$since one A spermatogonium produces 30.45 spernatids/spermatozoa aJainst the expected number of 64. Key words :
spermatogenesis, quantitative spermatogenesis, efficient of spermatogenesis, degeneration of sperma9: ogenlc cells, rams
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THERIOGENOLOGY INTHCkDU~TION Spermatogenesis is a continuous process taking place in various cellular stages which follow each other in time. Each stage consists of one or two generations of spermatogonia, spermatoc tes, and s rmatlds in addition to Sertoli cells. OnPy a limltegenumber of spermatogcnic stages and sub-stages could be seen in a cross section of a tubule. Based on meiotic divisions and the position of firstgeneration(more mature) spermatids, 8 stages of SEC have been identified In several mammals including rams(l-10). The relatively long-duration SEC stages have been subdivided in bull (11) and buffalo (12); identification of this sub-division has increased the specificity of this classification method, and consequently, the quantitative spermatogenesis (13) and cytochemical changes in spermategenie cells could be studied more precisely (14). In contrast to this classification method, the developent of acrosome and spermatid nucleus during spermatogenesis as seen in periodic-acid Schiff-haematoxylinstained preparations has been employed to study the efficiency of spermatogenesis in bulls (15) and gpats (16). With the processes described above,the present study was undertaken to sub-divide the already known eight stages of SEC identified in haematoxylin-eoslnpreparations in rams. The efficiency of spermatogenesiswas worked out by counting the different spermatogenic cells In %hese stages/ sub-stages. MATERIALS AND METHODS The left testis was obtained from 17 sexually meture (20 yr) rams (Ovis aries) The tunlca albuginea was removea and placedYiiXo-Z-7X9$saline solution; two or three small pieces (A-l.5 cm3) from each testis were fixed for 24 h in freshly prepared Zenker's fixative containing 10% formalin. The tissue was washed in running ta water for 24 h dehydrated treated for removal of ioil ine,a%ziembedded in paraffih wax (M.P. 58-60%). The sections were cut at 5 p and stained with baematoxylineosin (17).
486
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The meioticdiviiions,morphology and position of spermatids of the first generation were used to define eight main sta es of SEC. %ed. In the subStages l,2,3,4 and 8 were sub-divi stages of a given stage, the spermatogonia were of the s.ametype; however, other cell generations varied to differ%@g degrees. The relative occurrence of various stages and sub-stages was investigated by identifying the stage of the cycle in 2850 round tubular cross sections selected at random from different animals with at least 100 tubules per animal. From this, the rcentage of frequency was calculated.For the quantifs" ative behaviour of various spermatogenic cells, whole nuclei as well as their identifiable fragments were counted at each stage in at least 80 round seminiferous tubules. The dimensions of each cell type were recorded with the help of stage micrometer and occular micrometer. The counts of various germ cells were corrected for nuclear diameter, which may vary with thickness of a sectian. The correction was done by Abercrombie's formula (18) as outlined by Clermont and Morgentaler (13). For each cell type, the arfthmetic mean and standard error were calculated in each stage. The small fragments of Sertoli cell nuclei were not counted. Therefore, their number was not adjusted for nuclear diameter. FlESlJLTS
The salient features of various stages and substages are described below. The morphology of various spermatogenic cells was basicall, similar to that described already for the ram (25 . Eut in the present study based on the nuclear size and shape, zy o;t.;; and diplotene could be divided into their earlya n% phases; leptotene consisted of leptotene roper and granular lcptotene, which were similar to Phose in bull (11) and buffalo (20). Additionally, three generations of type A spermatogonia were distinguished in our study, in contrast to only two such gener t+z;zo;;ported by Ortavant (2). The nuclear dimensions oa spermatogenic cells are given (Tables 1 and 2). The descri tion for the morphology of cells is given briefly only wRere it is required.The distribution of various cell types and the comnosition of various stages/sub-stages are likewise summarized (Table 3).
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Table
1.
Variations in spermatogonial clear diameter ( p 1 and volume ( p ? in
ram Spermatogonia
Stages
Diameter
Volume
1-8
8.05
273
3b-4 2 g -la lb
7.40 8.23 8.00 8.50
% 291
z;.
3x 406
2a 2b
7908 7.44
186 216 136
223 Xn
4
6.80
165
B1
7
6.48
142
B2
8a-la
6.13
120
Spermatogenic
Stages
Stage 1. From the liberation of spermatozoa into the tubular lumen until the appearance of leptotene s ma tocytes (Plate 1, F’igures 1-3). This stage was c r aracterized by the presence of only round spermatlds near the tubular lumen. Stage la. From the liberation of spermatozoa into the tubular lumen until the end of type B2 spermatogonia (Plate 1, Figure 1). Stage lb. From the end of mitosis of type B2 rmatogonia until the end of the preleptotene f IF age (Plate 1, Figure 2). until
488
Stage lc .F’rom the end of the peleptotene the appearance of leptotene (Plate 1 ,Mgure
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1986VOL. 25 NO. 4
THERIOGENOLOGY
Table 2. Variations in germ cell nuclear diameter (p) and volume ($1 in rams
ceutype
Diameter
Volume
Spermatocytes Preleptotene Leptotene Granular leptotene Zygotene 1 Zygot3ene 2 Pachytene 1 Pat hytene 2 Diplot ene 1
5.69
6.13 6.39 6.83 ;:!4
8.31
89%
Secondary Diplotene spermatocyt&. 2 9.652
97
127
137 1
!% :I6 303
iii'
14z
S permatids 1. Spermatids (Stages 4,s) 2. Spermatids (Stages 6,8)
5.08
69
50%
90
Diplotene 2/ A, Spermatogonium (dtage lb)
1 *OS
1 loo
Diplotene 2/ 1 Spermatid
:*$?I .
MI
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THERIOGENOLOGY Table 3. The percentage frequency of stages of SEe and their cellular composition in the rams Stage la lb Ic
3 frequency
Sperma-, b permatocytes% rmids togonia 1 oung UQ Y%$-C
III
6 45 1 1;:;: . j 22.23
!"5;:j 10.78 .
A2 A2
GL z1
3a 3b
'6'*5;] 17.60 .
A3
=2 Z2
4a 4b
';*G?',10.61
In
z2
A3 IV
l
4.35
5
D, D,
D;!‘D, Ed SP
4.35
Ifi
c1
6
12.67 12.67
In
*?
7
11.03 11.03
B1
*1
El El
II R
Ed
R
Ed
R
Ed
R Ed B2V pl R Ed PI B2 A snermatoaonia were also present in all stages 1. eficeptin SEages 2a and 2b.II. PL, preleptotene; L,proper leptotene; GL,granular leptotene; Z,,early zygotene; Z2,1ate zygotene;P ,early pachytene; P ,late pachytene;D early diplotene;$2, late diplote%e; DI,diakinesis;ah-rI,secondary spermatocytes. III R round spermatids;El,elongatingspermatids;Ed, eiongaoed spermatids. IY. Some Xn spermatogonia also started appearing in stage
65*?5{ 11.73 .
2 4r
”
v.
490
$&matogonia B2 very rarely divided even ih Stage 8b to form preleptoLene spermatocytes.
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1986VOL. 25 NO. 4
THERIOGENOLOGY
titage 2. Prom the appearance of granular in grains,’ until the leptotene, i .e . lleptotene appearance of zygotene (Plate 1, Figures 4,5). The chromosomes of primary sperma W* ocy es at the leptotene stage were leading to the formation of ‘leptotene in grains’ due to the appearance of numerous granules in them. The older primary spermatocytes, which were previously a t the pachytene stage , were now in the early diplotene stage. The nuclei of spermatids began to elongate (Plate 1, Figure 4). Sta e 2b. Some primary spermatocytes had e early zygotene whereas older spermatocytes enteree were at the early diplotene stage. The nuclei of elongating spermatids were oriented towards the basal membrane of the seminiferous tubule (Plate 1, Figure 5). Some primary spermatocytes, which were previousV+ y a the late leptotene or early zygotene stage, had now advanced to the late zygotene stage.Tbe older primary spermatocytes and spermatids continued to increase in size (Plate 1, Figures 6,7,15). All the primary s permatocytes previously at the v*ear y zygotene stage advanced to the late zygotene stage. The older primary spermatocytes remained at the early diplotene stage. The nuclei of elongating spsrmatdds grouped in bundles and became embedded in Sertoli cell cytoplasm (Plate 1, Figure 6). Sta e b. The older spermatocytes increased in size t+ ecome the late dlplotene spermatocytes. The elongated spermatids attained their maximum length( Plate 1 ,Figure 7). Stape 4. Eegan with late diplotene/diakinesis spermatocytes and proceeded until the end of both meiotic divisions (Plate 1, Figures 8,s).
the
Stage 4a. The primary spermatocytes after a very short diaklnesis or late diplotene stage had already divided. The present stage ended \!ith the disappearance of prLmary spermatocytes that were earlier at the diplotene or diakinesis stage (Plate 1, Figure 8 > .
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THERIOGENOLOGY
Stage 4b. This stage was characterized by the absence of primary spermatocytes at the pachytene, diplotene,and diakinesls s ta es. Consequently, the primary spermatocytes of the $i rst, or older, were absent .The secondary spermatocytes generation and the figures of the second maturation divisions were present (Plate 1, Figure 9). The nuclei of young spermatids were incomp etely developed and possessed the grains v* of chromatin connected together by a fine thread.The younger spermatocytes, whichwere in the late zygotene stage, progressed i&o the early pachytene stage (Plate 2, Figure 10). Stage 6. The nuclei of young spermatids increased in size. Their chromatin of fine granules contained 3 or 4 karyosomes (Plate 2, Figure 11) . The bundles of elongated spermatids migratW* owards the lumen of the seminiferous tubules(Plate 2, Figure 12). Stage 8. F’rom thr; complete bordering of the seminiferous tubule with the elongated spermatids (spermatozoa)until the liberation of s ermatozoa into the lumen of the seminiferous tubule ( %1ate 2,F’igures 13,141.
The elongated spermatids eompletely border%%??* umen of the seminiferous tubule as Immature spermatozoa (Plate 2, Figure 13). elongated spermatlds were progressthe lumen of the seminiferous spermatozoa (Plate 2,Figure 14). Percentage tiequency of Stages of SE0 In the first four stages (containing one generation of spermatida percentage frequency was 61 .12$ compared to 3 in the last four stages(contalning two generations of spermatids)(Table 3) .This finding was similar to that in rams(21), bulls (11) and buffalo (121,but dlsslmllar to that of rats(l). Spermatogenlc Cell. Counts The dimensions of the nucleus in different germ cells are glventtables 1 and 2).The average number of SperaratogenM and Sertoll cell nuclei at various stages of SE and the number of tubules enamined for each stage follow(Table 4). 492
APRIL1986 VOL.
25 NO. 4
OLl OZl
W1
OCL
pa
:o9t
06
g*zTg*sz
L’L+Q’ZL
9’ lZZ.OZ z*l~Z’oz
$:!%:I
OL 91 -“1
92 =f 92 w
$
8:oz8:8
98
=8 L 9
THERIOGENOLOGY EXPUNATIONOF FIGURES A part of seminiferous tubules show the cellular associations in different stages of the seminiferous epithellal cycle In haematoxylin-eosin stained sections from a portion of testes of rams (X 1080; Figures ,l-14; X 2160, Figure @>.The lumen of each tubule is on right hand side. PLATEIa Figures
Stages la, lb, and lc of cycle differ from one another in having B spermatogonla,prelepto&ene a&
l-3.
spermatocytes respectively towards periphery. The most characteris tls
the
feature of these stages is the presence of only one generation of round sperm+ tids. Figures 4-T.
Stages 2a and 2b which differ in showing granular leptoteneand early zygotene phases of second generation of spermatocytes; the first generation of spermatocytes has advanced to early dlplotene.The elongation of spermatlds starts in stage 2a and continues in stage 2b.
Figures 6-7
l
Stages 3a and 3b differ in the presence spermatids and the elongaof elongatin ted spermatl %s which attained maximum length .The first generation of spermatocytes from early diplotene In stage 3a advances to late dlplotene In stage
3b.
Figure 8.
Stage 4a shows metaphase,anaphase telophases of meiosis-I.
Figure 9.
Stage 441characterized
aLnd
by the presence of secondary apermatocytes ,meiosis-II and young spermatlds and by the absence of primary spermtocytes of first
generation.
lanation aExp
494
of abbreviations follows Plate 2.
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1986VOL. 25 NO. 4
THERIOGENOLOGY
Plate 1
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THERIOGENOLOGY
Figure 10.
Stage 5 showing primary spermatocytes advanced to early pachytene,the young round spermatids with incompletely developed nuclei are preseti throughout the seminferous tubule in addition to first generation of elongated spermatlds
Figure 11.
Stage 6 showing nuclei of round spermatids with increased size;the other cell types being similar to those in stage 5.
Figure 12.
Stage 7 characterized by presence of B, spermatogonia and the movement of bundles of elongated spermatids towards the tubular lumen.
Figure 13.
8a characterized by the bordering of tubular lumen by immature SpermatoWa. B spermatogonia are present.Residual bgdies are also seen. Stage 8b characterized by the presence of immature spermatozoa at isolated pla.ces. The other cell types are similar to those of stage 8a.
Figure 14.
Figure 15.
Stage
k spermatogonia In association with late diplotene and late zygotene, elongated spermatids of stage 3b.
aFxplanation of abbreviations follows Plate 2.
496
APRIL 1986 VOL. 25 NO. 4
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THERIOGENOLOGY
Plate 2
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THERIOGENOLOGY
Abbreviations in plates different types of A spermatogonia different types of B spermatogonia early diplotene late di_plotene elongated spermatids EL
elongating speWids
GL
granular leptotene
In
intermediate spermatogonia
L
leptotene
W-I
figures of meiosis-I
M-II
figures of meiosis-II
PI
preleptotene
p1 P, L
early pachytene late pachytene
EB
residual bodies
RS
round spermatids
S
Sertoli cells
ss
secondary spermatocytes
SZ
spermatozoa
YS
young round spermatids
z1
early zygotene
z2
498
late zygotene
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THERIOCENOLOGY
A type spermatogonia bad slightly elongated nucleus conta1ning fine dust-like cbromatin and were cbaracterlzed by the presence of only one nucleolus. These cells were missing only in stages 2a and 2b of the cycle. An average of 2.0 type AI spermatogonia were present in Stage lb which became 3.9 (Stage 2a) and 7.1 (Stage 3a), showing approximately twice the increase in their number in each of these stages. This indicated the occurrence of two mitotic divisions of t e A spermatogonia during these stages. The count of ce9Ps suggested that the major part of these mitotic divisions occurred between Stages lb-lc and 2b-3a. The combination of morphological observations with the counting studies suggested that all Al spermatogonia divided to form A2 s ermatogonia which subsequently divided to form a majcrley of A spermatogonia (Plate 2, Figure 15) and some Al spermatogon?a. 4 spermatogonia could be distinguished from the parent A spermatogonla because they were smaller in size snd they usually contained more than one nucleolus. Compared to A spermatogonia showed a relatively dark2 spermatogonia of stage 3a were onla but in stage 3b they increased in size (Table 1). A, spermatogonia showed a progressive increase in their size from Stage 3 until the end of the cycle(Table 1); boweoer,tbeir number decreased from 2.0 to 1.5 (Table 4), demonstrating a 25% degeneration of A type spermatogonla during these stages. In the second half of the cycle, the nuclei of these cells fragmented, and the celkultima~ely degenerated. Subtraction of A type spermatogonia in Stage 4a(A > from those of Stage ja (A +A > iridicatedthat 5.1 of Ai spermato:onia produced 8.2 IJIspermatogonia in Stage 4b. The assumption is made that the number of A spermatogonia that did not divide to form In spermatogonia remained constant in Stages 3 and 4. The total number of A type spermatogonia in Stage 3b was not considered because some In spermatogonia also appeared in this stage. Furthermore, In spermatogonia contained more darkly stained nuclei with coarse chromatin in their centers and on their nuclear envelopes than A spermatogonia. The nuclei of In spermatogonia te& ed to be smaller, more spherical& cohtained more than one small nucleolus. Although the number of In spermatogonia was maximal in Stage 4b , this sum was
APRlL1986VOL.25NO.4
499
THERIOGENOLOCY
less than twice the number of the parent 1:type cells (Table 4). Thus, a 13.7$ degeneration of A spermatogonia was shown before the In spermatogonia 3ere formed. The number of In spermatogonia remained almost constant throughout their existence until Stage 6 suggesting the existence of only one generation of these spermatogonia in the SEC. In Stage 6, 8.8 In spermatogonia formed 12.8 El spermatogonia, the number of which was also less than twice the number of the&r parent cells. This indicated a 2'7,3$ degeneration of In spermatogonia, In the beginning of Stage 8a,the Bl spermatogonia divided to produce 20.2 spermatogonia.This number was again less than twice the number of parent cells, suggesting a degeneration rate of 21.2%. The division of RI type sT:ermatogoniato form a second type of F spermatogonia was coupled with morphological observations that showed the existence of two generations of 1?spermatogonia, Bland B . The nucleus of B in contrast to In type spermat5 gonia, tended to bg'smaller, more spherical and more darkly stained; it also contained a greater number of nucleoli which were smaller.The more darkly stained nucleus was due to the increased amount of chromatin crusts. B spermatogonia generally divided at the end of Stage la, o!$ very rarely at Stage 8b, to form twice the number of primary spermatocytes that showed a constant number throughout the various @ases of meiosis at all the stages of SEC. This suggested that the second generation of B spermatogonia and primary spermatocytes do not degenerabe. In Stage 4a, 78.5 secondary spermatocytes were produced. This number was almost twice the number of primary spermatocytes indicatin no degeneration of spermatccytes had occurred during firsI meiosis. To count the number of secondary s~r-~tocytes _t~~bn?eswith only secondary spermatocytestrare situation) or secondary spermatocytes with dlplotene or diakinesis (Stage 4a) were considered.After adjusting for the number of nuclei of these primary spermatocytes, their number was doubled to get the number of secondary spermatocytes; it was assumed that spermatocytes did not degenerate during these divisions. From 78.5 secondary spermatocytes in Stage 4b, l!?% Step 1 spermatids were produced. This showed that there was no degeneration in meiosis-II. The number of round sperm&ids during SEC apparent1 was not constant* the small increase or decrease in tK eir number did not follow
500
APRIL1986VOL.25NO.4
any regular trend and suggested that these small variations in their number could be due to counting errors and not to degeneration. The number of Sertoli cells was constant throughout SEC (Table b),suggesting that there was no division in adult testis. Throughout the various stages, their number could not be adjusted for differences in nuclear diameter because of the changing shape of the nucleus within the varloua stages. The nucleus lay along the basement membrane forming a triangle with it in Stages 8b to 2b(PLate 2, Figure 140 Plate 1,Figures I-5). During the detachment of elongated sperrratidsfrom the Sertoli cells in Stages 6 and 7 (Plate 2, Figures 11,12), some of the nuclei took on a spindle-shaped appearance while most of them were flattened against the basement membrane. DISCUSSION Eight sta es of SEC have also been reported in some mammals inclu%ing rams (see references in the Introduction). But the subdivision of various stages with relatively long duratlon(Stages 1 ,2,3,4,8) have been previously done only in bull (11) and buffalo (12). If each sub-stage in rams were raised to the level of a stage, the total number Eventien subof stages in a cycle would be 14. divisions were based on the development of acrosomes requiring additional stages, only 12 stages have been reported in bull (15); however, using the outlined procedure, we have reported 14 stages in buffalo (22) and goat(l6). AS in our present study on rams, the Sertoli cells in other species have also been reported to undergo durin SEC and these cells have variations in nuclear sha also not divided in the aze ults (If ,15,16,23).
Due to the occurrence of six peaks of spermatogonial mitosis in SEC stages, we were able to identify six generations of spermatogonia. Out of these,three generations A ) one of In spermatogonia, were of A spermatogonia (A and two of B spermatogonia'1BFh2)? &eviously , only two generations of A sperma_togonia~A1,&?) and a total of only five generations were reported in rams (13 and in bulls (3). In contrast, there were six spermatogonial generations in bull (ll), buffalo (l3,2O), and goat (lG).The number of generations of various spermatogonia varies with the species studied and with the investigator(2k).
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THERIOGENOLOCY
The cell-specific degeneration observed in this study has also been re orted for certain cell types in some mammals (23,2$), but the systematic study of the behaviour of all spermatogenic cells has been done only in bull (I51, buffalo (13),md goat (16). The present observations have shown 25, 13.7, 27.3 and 21.25
degeneration
of types
A
A
In and R
spermatogonia, respectikely, 1; r&s.Calcula%ed from the data of Eerndtson &:Desjardins (15),these cells show 33, 6.3, 27.8 and 16.2$ degeneration rate in bull. In buffalo, these cells show 20, 18.75, 10.2 ard 15.5% degenera ion, respectively (13) compared with 26.3, 15, 25, 25.8k degeneration of corresponding cells in goat (16). As in the cited studies no degeneration could be observed throughout the meiotic popbase in mice (25), rats (26,27), bulls (3,15,28) and buffalo(l3). In our study, no degeneration was observed during the division of secondary spermatocytes to form spermatids. In contrast, there was a 18.7, IO and 10.4% de eneration of secondar spermatocytes respectively in bullf15), buffalo (133;, and goat (16). The deleterious effects of long daylight photoperiod on some spermatogenic a cells in rams have been studied (29). This study reported 19 and 23$~degeneration during the divisions of A and In spermatogonia, in addition to 36 and 4% degenkations, during earl pachytene and reduction ~~:Pz%l us the extene of degernration in rams differs according to normaland long daylight conditions. Due to their elongation and uneven distributioqthe number of spermatids could not be counted after Stage lc,Because of chromatin condensation and other some spermatids may degenerate ;~~;~e';"~~g$;g~~ 8 as has been reported for other species (26). The extent of degeneration that occurs at various steps of spermatogenesis becomes clear by the mean number of spermatids produced per A spermatogonium available for division following Stage 3a . This was only k7.58$ of the number expected inthe absence of degeneration. This number has been re ortdd to be 43 43.32,znd 33.1s in bull (15) buffalo ?13), and goat (16) respectively. If the possibility of degeneration during spermiogenesis was not considered, one A spermatogonium available for division after Stage 3b praduced 30.45 spermatozoa in rams instead of the expected number of 64. One A3 spermatogonium has been reporter?to produce 27.73,
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28 and 21.1 spermatozoa in buffalo (131, bull(l'j)and
goat(16) respectively. A spermatozoal loss has been reported in bulls during trans rt through the . It is concluded, epidldymis and vas deferens (30p" therefore that based on an ei ht-stage classification, the sub-division of sta es wi!h relatively long nvestigating the differentiaduration is useful for f: tion of spermatogenic cells. Our study has also found that spernatogeaesis in the ram is more efficient than that In the bu11(15), the tiffalo(l3) and the goat(16).
1. Roosen-Runge, E.C. and Giese1,L.O. Quantitative studies on spermatogenesis in the albino rat.American Journal of Anatomy & : l-30 (1950). 2.
Ortavant,R.Le cycle spermatog&&ique chez le b&er. D.Sc. Thesis. University of Paris,Paris(l958).
3. Amann, R.P. Reproduction capacity of dairy bulls.IV. Spermato enesis and testicular germ cell degenerafion. American Journal of Anatomy 110: 69-78(1962). 4. Hochereau,M.T. Constance des frequences relative des stades du cycle de 1' epithcyliumseminifere chez le taureau et chez le rat.Annales de Biolo e animale,Biochimie,Biopbysique 2: 93-102 (1963E
5. Swierstra, E.E. and Foote, R.H. Cytology and kinetics of spermatogenesis In the rabbItJournal of Reproduction and Fertility & 309-322(1963). 6. Swierstra,E.E. Cytology and duration of the cycle of the seminiferous epithelium of the boar;duration of spermatozoon transit through the epididymis. Anatomical Record m:171-186 (1968). 7ASwlers tra, E.E., Gebauer M.R. and Pickett,B.W. Reproductive physloiogy of the stalllon.I.dpermatogenesls and testis composition.Journalof Reproduction and Fertility 9:113-123 (1974) 8. Tiba,T. Ishikawa,T. and Murakomt,A.Hlstologiche u n&ersuchung der kinetik der spermatogenese beim Mink (Mutele vision .I&emenepithelzyklus in der Paarungsze 1G;J1apanese Journal of Veterinary Research 15:73-87 (1968).
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9.
Foote,R.H.,Swierstra,E.E.andRunt,W.L.Spermatogenesis in the dog.AnatomicalRecord u:341-352(19729.
?O.Schuler, H.M.and Gier,M.T. Duration of the cycle of seminiferous epithelium in the prairie vole (Microtus ochronaster ocbroaaster).Journalof Experimental Zoology,?q2:1-12(?976). ?l.Bochereau-de-Reviers,N.T.Etudes des divisions spermatogoniales:etdu renouvellement de la s ermatogonie souche chez le taureau.D.Sc. tResis,University of Parls,Paris (1970). l2.Guraya S.S.& Bilaspuri,G.S. Sta es of seminiferous uration of epithelial cycle and relative 6: spermatogenic processes in the buffalo.Genenbaurs or hologisches Jaharabuch Leipzig 122:147-161 19 6). ?? 13,Bilaspuri,G.S.and Guraya,S.ti.Quantitative studies on spermatogenesis in buffalo (Bubalus bubalis). Reproduction,Rutrition,De'veloppementm-982 (1980). 14.Bilaspuri,G.S.Morpbologicalhistochemicaland biocbemical studies on the testes of some farm animals. Ph.D. thesis Punjab Agricultural University, Ludhiam,Ind$a (1978). l!j.B@~?ndtson W.E. and Desjardins,C. The cycle of seminiferous epitbelium and spermatogenesis in the bovine testis.AmericanJournal of Anatomy. 140 :167-180 (1974). 16.Bilaspuri,G.S.and Guraya,S.S. The seminiferous epithelial cycle and spermatogenesis in goats (C r hircus).Journal of Agricultural Sciences, CZ%!d~RJ:368( 1984). 17.Humason,G.L. Animal Tissue Techniques.W.H.Freeman and Company, San Francisco (1967). 18.Abercrombie,M.Estimation of nuclear po lation from sections.AnatcmicalRecord&r 238~2@(1946). .Clermont,Y.and #orgentaler,H. Quantitative studies of spermatogenesis in the hypophysectomlzedrat. Endocrinology B:369-382( 1955). Spermatogenic cells and Bilaspuri,G.S. 20. Guraya S.S. of {be buffalo(Bubalus bub lis)testls.Indin Journal of Anlm~nces +368-375(1976!. _ 19
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.Ortavant,R.Spermatogeneslsand morpholo y of the spermatozoon.In:Cole,H.H.and Cupps,pi.T.(eds). Reproduction m Domestic Animals.Academlc Press,New York,l959,pp.l-50.
22.Guraya,S.S.and Bilaspuri G.S.Stages of seminiferous epithellal cycle in the buffalo.Annales de Biolo le animale,E%ochimle,Blophysi~ue 16: ,3791$4 (1976). 23.Courot M., Hochereau-de-Reviers,M.T.and Ortavant R.Bpermatogenesis. ns Johnson,A.D. Gomes,W.R. and Van Demark N.L. 5 eds.).The Tes&,vol.I, Academic Press,New York, 1970.~~ 339-432. 24.Clermont,Y. Kinetics of spermatogenesis in mammals: Semidf&~us epithellum cycle and spermato onlal renewal. Physiological Reviews &:198-236?1972). 25.0akberg,E.F.Sensitivltyand time of de eneration of spermatogenetic cells Irradiated in various stages of maturation In tht:mouse.. Radiation Research _2:369-391( 1955). 26.Clermont Y.Cuantltative anelysls of s rmatogene&s of the rat: A revlaed model for tI? e renewal of s ermato on1 .American Journal of Anatomy mt l&-,29( f962?. 2c/.Desclln,J,andOrtavant,R. Influence des hormones gonadotropes sur la duree des processus spermatog&&iques chez le rat.Annales de Blolo le anlmale, Biochimle,Blophysique2: 339-&k (1963). 28.Attal,J. and Courot,M. De'veloLxnent testlcularie et Establishment de la spermatogenese cheze le taureau.Annales de Biolo le animale,Biochemie, Biophyslque 3~219-241 (1663). 29.0rtavant,R. Action de 13 dure'edleclairemen;tsur les processus spermatogenetiqueschez le belier. Competes Rendus des Seances de la Soclete' de Biologie et de Ses Finaliales =:471(19563. 30.Amann,R.P. and Almquist J.O. Reproduction capacity of dairy bulls.VI.Ehfect of unilateral vasectomy and ejaculation frequency of sperm reserves; aspects of epididymal p4ysiology .Journal of Reproduction and Fertility 1:260-268(1962).
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