the jelly envelopes and fertilization of eggs of the newt, notophthalmus viridescens

The Jelly Envelopes and Fertilization of Eggs of the Newt, No toph tha lmus viridescens

ELLEN W. MCLAUGHLIN Department of Biology, Emory University, Atlanta, Georgia 30322, U.S.A

AND A. A. HUMPHRIES, JR.

ABSTRACT Fertilization in Notophthalmus viridescens is internal and involves passage of the sperm through five layers of egg jelly (55-51, from outermost to innermost), each of which is secreted by a discrete region of the oviduct. Polyspermy is normal. Passage of the sperm through the jelly and into the egg was studied by a technique of artificial insemination similar to natural insemination, in that undiluted fluid from the vas deferens was applied directly to eggs with various layers of jelly present, followed by flooding with water three to five minutes later. In general, successful fertilization increased as the number of jelly layers increased; jellyless coelomic eggs were not fertilizable. Sperm passage through the jelly and into the egg usually occurs within one to three minutes. Upon hydration of the jelly, barriers to sperm penetration develop in layers 55 and 53. Changes in the egg jelly thus seem to be involved in the restriction of polyspermy to a low level.

The fact that the jelly envelopes of amphibian eggs are important for fertilization is well established. Various investigators have shown that jellyless eggs are generally not fertilizable or that fertilizability is related to the quantity or quality of the jelly present (see Elinson, '73, for references). Despite the rec- ognition of the significance of these envelopes, however, there is as yet only limited under- standing as to just how they are involved in fertilization.

The egg jelly of Notophthalmus is not a ho- mogeneous, amorphous mass, but is made up of a series of layers of contrasting composition and morphology deposited about the oocyte as i t moves through an oviduct differentiated into regions producing several more or less dis- tinct secretions (Humphries, '66, '70; Humph- ries and Hughes, '59; Humphries et al., '68). Although the nature and number of the layers are not the same in different species of amphibians, some features seem to be common to many species of anurans and urodeles. Beyond the general statement that the envelopes are mucopolysaccharide and glycoprotein in composition, there is evidence from various in- vestigations that the innermost envelope contains sulfated mucopolysaccharide (for references see Hedrick et al., '74). Anoth- er commonly reported component is sialic acid (Bolognani et al., '66; Humphries, '66;

J. MORPH. (1978) 158; 73-90.

Humphries et al., '68; Lee, '67; Freeman, '68; Pereda, '70; Martinez and Olavarria, '73; Yurewicz et al., '75). The complexity of the envelopes is considerable and it is reasonable to assume that enhancement of fertilization is but one of several aspects of jelly function, some of which may be of importance before fertilization and some only after development of the zygote begins.

In Notophthalmus viridescens the oviduct is differentiated from ostium to cloaca into five readily distinguishable regions designated A through E, each of which secretes one of the five jelly layers, 51 through 55 (Humphries and Hughes, '59; Humphries, '66). That is, eggs from the posterior part of region A have jelly layer 51, eggs from region B have layers 51 and 52, eggs from regions C, D and E have layers 51-53, 51-54 and 51-55, respectively. The marked zonation of the oviduct makes Notophthalmus particularly favorable for studies of this kind, since eggs can be removed from the various regions with sure knowledge as to which of the jelly layers are present.

In order to learn more about the natural course of events following deposition of the sperm on the jelly envelopes and to attempt to clarify the significance of these several layers in fertilization, we undertook an investigation

' Present address: Department of Biology, Samford University, Birmingham, Alabama 35209, U.S.A.

73

74 E. W. MCLAUGHLIN AND A. A. HUMPHRIES, J R

of the sperm penetration process in the urodele Notophthalmus viridescens using artificial insemination followed by cytological studies.

In the natural situation, insemination of Notophthalmus oocytes occurs in the cloaca from a supply of sperm stored in the spermatheca (Kingsbury, 1895) and fertilization is normally polyspermic (Fankhauser and Moore, ’41). Eggs a re usually inseminated one at a time and deposited individually a few minutes afterwards. Ordinarily the female attaches each egg to vegetation such as Elodea, often by wrapping the egg in a leaflet of the plant. The methods used for artificially inseminating anuran eggs have not proved to be very effective for these animals, probably because fertilization is normally internal. For urodeles, the most successful method has been tha t involving the use of a concentrated sperm suspension (Hadorn and Fritz, ’50, for Triton alpestris). Dilute suspensions, such as those employed by Hughes (’56) wi th Notoph- thalmus viridescens, are considerably less effective. Since there was no established method for obtaining a high percentage of fertilized eggs of this species through artificial insemination, the development of a satisfactory method was one of the early tasks of our investigation. The major part of the study, however, was centered in an effort to relate fertilizability to the presence of various jelly layers and to investigate in several ways the movement of spermatozoa through the jelly and into the egg. A considerable amount of work has been done showing the relationship between the jelly layers and sperm penetration in anuran amphibians (see Elinson, ’71, for references), but very little is known of the situation in urodeles. Other than the recent work of Picheral (’77); there is little information about the cytological events associated with sperm passage through the jelly in either anurans or urodeles.

MATERIALS AND METHODS

Adult specimens of Notophthalmus viridescens were collected near Franklin, North Carolina and in eastern Tennessee and kept in a refrigerator a t 12°C until needed. Ovulation was induced by intracoelomic injections of 170 IU of gonadotropin (Antuitrin “S”, Parke- Davis) every three days until egg deposition commenced.

For experiments requiring oocytes from the

oviduct, the animals were injected until egg laying started and then the oviducts were removed and placed in covered petri dishes lined with moist filter paper. At no time were the oviducts immersed in liquid. Eggs were removed from the oviduct by gently tearing the wall along i ts length with watchmaker’s forceps. Eggs from regions B, C, D, and E, which had firm jelly capsules, were left in the petri dishes until used, while those from region A, which had little or no jelly support, were placed in a small drop of amphibian Ringer’s solution until needed. Coelomic eggs were treated in the same way as those from region A.

Since ovulation and egg deposition occur over an extended period, the number of eggs in the coelom and oviducts a t any time is usually small, with the largest accumulation occur- ring in oviduct region E. When large numbers of coelomic eggs were required, therefore, they were obtained by ligating the anterior portion of one oviduct so tha t eggs tended to accumulate in the body cavity on tha t side. Animals were allowed to recover from the surgery for two weeks or more before ovulation was induced. Jellyless eggs from region E were prepared by immersing the eggs in water for five minutes and then gently forcing the egg out through a tear in the swollen jelly layers.

Techniques of artificial insemination Dilute sperm suspensions were prepared by

macerating one vas deferens or testis in 10 ml of amphibian Ringer’s solution followed by dilution prior to use (1: 10 sperm:Ringer’s). Eggs were immersed in 10 ml of the 1:lO sperm suspension which was replaced 5 to 15 minutes later by standing tap water. Standing tap water was used as the culture medium for all inseminated eggs and developing embryos. In experiments using undiluted sperm suspensions, care was taken to keep the eggs free from contact with fluids prior to insemination; for these, portions of the vas deferens or testis were excised and their contents were applied directly to the jelly surface. Three to five minutes later the eggs were flooded with water and maintained until results could be recorded.

As will be reported, the technique of inseminating eggs with a n undiluted sperm suspension t aken directly from the vas deferens proved to be superior to the others tested, thus in all subsequent experiments this method was used to inseminate eggs from

JELLY ENVELOPES AND FERTILIZATION 75

the five regions of the oviduct. Jellyless eggs were inseminated in the same way as eggs from the oviduct but only after the Ringer’s solution had been removed. After insemination, all eggs were maintained in tap water for about 15 hours. At this time, the number of cleaving eggs was recorded and these were usually allowed to develop until hatching occurred. Uncleaved eggs were fixed, sectioned and stained for cytological examination. In all experiments involving artificial insemination, region E eggs were used as controls to test the fertilizing capacity of the sperm prep- aration.

Methods for studying sperm penetration The ra te of sperm penetration through the

jelly capsule was studied by rapid freezing of region D and E eggs a t 1, 3, 5 or 10 minutes after insemination without exposure to water. At the appropriate time, eggs were immersed in a beaker containing 2-methyl bu tane chilled with liquid nitrogen. After freezing, some of the eggs were transferred to test tubes and kept for five to seven days in 100% ethanol cooled by an acetone-dry ice mixture; others were fixed for two days in 0.001 M cetyl- pyridinium chloride (CPC) in 10% formalin chilled to 5°C. After sectioning and staining, the eggs were examined for the presence of spermatozoa in the jelly layers and cytoplasm.

A limited number of studies of fertilizaion were made using living material and phase contrast microscopy. For these studies, microscope slides with a bored perforation 6 mm in diameter were used. One side of the hole was covered with a cover slip, sealed with wax and the egg placed in the chamber and inseminated. After covering the upper side of the hole with another cover slip, observations were made with a n inverted phase contrast microscope fitted with equipment for flash photography. The relative opacity of t he outer jelly membranes, especially J5, makes study of all but superficial aspects of fertilization of fully jellied eggs impossible, but mechanical removal of t he outer layers or use of partially jellied eggs from the oviduct makes observation of sperm penetration feasible. Using such partially jellied eggs, preparations were studied before, during and following hydration of the jelly layers. For this method we are indebted to Doctor Jorge Raisman, of t he Uni- versity of Tucuman, Argentina, who first dem- onstrated i ts feasibility during a stay in our laboratory.

Treatment of eggs immersed in water In one series of experiments, eggs from re-

gions D and E were immersed in water for 15, 30 or 60 minutes prior to insemination. At the end of each time interval, t he eggs were drained, inseminated, reflooded with water and then maintained until the number of cleaving eggs could be recorded.

Fixation and processing

Fixatives were selected so as to facilitate the study of several different cytological features. Eggs were fixed in either Bouin’s picroformol for nuclear and cytoplasmic struc- tures or in CPC-formalin for study of the relationship of spermatozoa to the jelly layers or egg. Slides were triply stained with Harris’ haematoxylin, fast green and the periodic acid-Schiff procedure (PAS).

OBSERVATIONS AND RESULTS

The five jelly layers of the newt egg, to which reference will be made in the following descriptions, are shown in figure 1. Also present is the incipient capsular chamber, a fluid filled space tha t begins developing between layer 52 and the vitelline membrane while the egg is still in the oviduct, but which reaches i ts full size only after t he egg is immersed in water. The spermatozoon, made up of a head about 150 pm long, a neckpiece of 37 pm, and a long tail (about 400 pm) with a conspicuous undulating membrane (Baker, ’661, is normally deposited on layer 55.

Insemination technique When eggs from region E were artificially

inseminated, the best results were obtained with a n undiluted sperm suspension from the vas deferens smeared directly on the jelly. As indicated in table 1, about 69% of eggs taken from region E cleaved when artificially inseminated by this method. Development of eggs tha t cleaved was normal through hatching, at which time observations were termi- nated. Insemination with spermatozoa from the testis was unsatisfactory, with only 1 of 36 eggs cleaving. Eggs inseminated with a 1 : l O sperm suspension from the testis or vas deferens gave no evidence of fertilization.

Twenty region E eggs inseminated with an undiluted sperm suspension from the vas deferens, but uncleaved after 15 hours, were examined cytologically; t he surface of the jelly was found to be covered with hundreds of

76 E. W. MCLAUGHLIN AND A. A. HUMPHRIES, JR .

TABLE I

Cleavage and fertilization in artificially inseminated eggs from the coelom and regcons A through E of the ouiduct

Source (coelom or Number Number Cleaving eggs Fertilized eggs I

region of of of oviduct) animals eggs Number Percentage Number Percentage

~ ~~~

Coelom 16 68 1 1.5

Dejelhed eggs 10 25 1 4.0

A 17 71 11 15.5 B 17 47 12 25.5 C 27 83 34 41.0 D 20 58 48 82.8 E 42 370 254 68.7

1 1.5

1 4.0 14 19.7 24 51.1 52 62.7 50 86.2

260 70.3

' Includes cleaved eggs and uncleaved eggs showing evidence of fertilization as indicated by sperm entry into the cytoplasm.

spermatozoa. All eggs had spermatozoa penetrating the jelly layers and, in 14 cases, spermatozoa had reached the capsular chamber. Only one of the uncleaved eggs showed any evidence of sperm penetration into the cytoplasm, and in this case several asters were the only indication. The observations seem to show clearly that failure of these eggs to cleave was due primarily to failure of the sperm to penetrate into the cytoplasm, rather than failure to penetrate the jelly layers.

No cytological evidence of fertilization was observed in serial sections of 21 uncleaved region E eggs inseminated with an undiluted sperm suspension from the testis. Although most of these eggs had many spermatozoa on the jelly surface, only occasionally were spermatozoa found penetrating the jelly layers. Cytological studies were also made of eggs inseminated with a 1:10 sperm suspension from the vas deferens. Although a few isolated spermatozoa were found on the 55 surface, none were observed in the jelly layers, capsular chamber of egg cytoplasm. Thus, in contrast to eggs inseminated with a concentrated sperm suspension from the vas deferens, failure to cleave in these cases seems due to inability of sperm to penetrate the jelly.

Comparisons between naturally and artificially inseminated eggs from the same animals revealed a higher percentage of cleavage in naturally inseminated eggs (67176, 88%) than in eggs artificially inseminated with sperm from the vas deferens (51181, 63%). Serial sections of naturally inseminated eggs showed very few spermatozoa associated with the jelly capsules. In the eggs examined, fewer than 30 spermatozoa were found on or in the jelly of each egg, with an average of six in the egg cytoplasm.

Artificial insemination of eggs from different levels of the ouiduct

In collecting data on artificial fertilization in eggs from different regions of the oviduct, both the number of cleaving eggs and the number of uncleaved eggs showing evidence of sperm entry into the cytoplasm were recorded. Since eggs in both these groups were considered to be fertilized, the two sets of figures have been combined to give the total number of fertilized eggs for each region of the oviduct. The results are shown in table 1. Data for coelomic and dejellied region E eggs are also included.

As reported above, cleavage was observed in about 69% of the eggs from region E having jelly layers 51-55 present. Eggs from region D with jelly layers 51-54 showed a percentage fertilization (86.2%) very much like that recorded for cleavage (82.8%). Relatively low percentages of cleaving eggs were recorded for regions C (41.0%) and B (25.5%), but when cytological examinations were made on uncleaved eggs, the percentage of fertilization, as indicated by sperm penetration into the cytoplasm, increased to 62.7% for region C and 51.1% for region B. Distributed throughout the cytoplasm of most of these uncleaved but fertilized eggs from regions C and B were numerous asters with associated chromatin (fig. 21, and almost half of the eggs sectioned had more than ten asters per egg.

Artificially inseminated eggs from region A showed low percentages of both cleavage (15.5%) and fertilization (19.7%). Eggs from the anterior portion of this region had not yet acquired a discernible jelly coat, whereas those from the posterior portion were en- veloped in an amorphous jelly layer which dis- solved and disappeared shortly after immer-


TABLE 2

Time required for sperm penetration through the jelly layers and into the cytoplasm of eggs from regions D and E

Number and percentage of eggs with spermatozoa

Region In jelly layers In egg of Fixation On jelly In jelly and capsular cytoplasm

oviduct time after Number Number surface only layers chamber (fertilized) insemination, of of

minutes animals eggs Number Percent Number Percent Number Percent Number Percent

E 1 6 59 11 18.6 48 81.4 13 22.0 1 1.7 3 8 53 1 1.9 52 98.1 38 71.7 24 45.3 5 8 50 0 0.0 50 100 46 92.0 26 52.0

10 6 44 3 6.8 41 93.2 41 93.2 26 59.1

D 1 5 12 0 0.0 12 100 12 100 2 16.7 3 4 11 0 0.0 11 100 11 100 5 45.5 5 3 13 0 0.0 13 100 13 100 9 69.2

sion in water. All the cleaving eggs initially possessed some jelly, but there were many eggs with visible jelly which were unfertilized.

Jellyless eggs, whether from the coelom or from region E, were essentially unfertilizable, although a single one from each group cleaved.

In summary, therefore, the results suggest that the fertilizability of eggs increases as they pass down the oviduct as far as region D, with region E having only a slightly lower percentage of fertilization than region D. Some jelly seems to be required for fertilization.

Sperm penetration through the jelly and into the cytoplasm

Eggs from regions D and E were frozen in- stantaneously at 1, 3, 5 and 10 minutes after insemination without exposure to water. After processing, serial sections were examined to determine the distribution of spermatozoa in the jelly layers, capsular chamber and cytoplasm. Results are summarized in table 2.

It is apparent that sperm penetration can be rapid, for a t the end of only one minute spermatozoa were found in the capsular chamber and cytoplasm of some region E eggs having all five jelly layers. For the most part, however, the spermatozoa observed in region E eggs fixed a t one minute were just beginning to enter the 54 layer. In eggs fixed a t 3, 5 and 10 minutes, spermatozoa were generally found in the egg cytoplasm as well as in the capsular chamber and all jelly layers. Although actual counts were not made, it was evident from observation of both living and fixed material that the number of spermatozoa in the capsular chamber increased with time. Observa-

tions of the living eggs with phase contrast revealed that spermatozoa in the capsular chamber usually continued to move for 15 minutes or more after the beginning of jelly hydration.

Sperm penetration is more rapid in region D eggs than in eggs from region E. The percentage of region D eggs with spermatozoa in the jelly layers, capsular chamber or cytoplasm after one minute is consistently higher than that for region E. The rate and extent of movement of spermatozoa into the 54 layer of region D eggs was remarkable. This layer was usually invaded by so many spermatozoa that they formed a dense network within the jelly (figs. 3, 4); there appeared to be little or no barrier to penetration. No such situation was observed in region E eggs with a comparable degree of insemination. In these eggs, only a relatively few scattered spermatozoa were found distributed throughout the jelly layers, while sperm were numerous on layer 55 (figs. 1, 5, 6). It was also noted that, regardless of fixation time, there was a greater accumulation of spermatozoa in the capsular chamber of eggs from region D than in that of eggs from region E.

Cytological observations of this material allow a description of the stages of sperm penetration through the jelly layers and into the cytoplasm. Immediately after artificial insemination, spermatozoa adhere to the sticky 55 layer; in stained sections they are usually seen lying parallel to its surface (fig. 1). Liv- ing preparations reveal, however, that sperm lie at various orientations to the jelly surface, that they are hoop-shaped, and that they are highly active. The tip of the sperm head is the first part to enter the 54 layer, moving a t an angle of approximately 90" to the egg surface.

78 E. W. MCLAUGHLIN AND A. A. HUMPHRIES, JK.

TABLE 3

Sperm penetration and fertilization in region D and E eggs immersed in water for 15, 300r 60 minutes prior to insernination

Number and percentage of eggs with spermatozoa

Region In jelly layers In egg of Immersion On jelly In jelly and capsular cytoplasm

oviduct time before Number Number surface only layers chamber (fertilized) insemination, of of

minutes animals eggs Number Percent Number Percent Number Percent Number Percent

E 15 8 42 42 100 0 0.0 0 0.0 0 0.0 30 6 35 33 94.3 2 5.7 0 0.0 0 0.0 60 4 52 51 98.1 1 1.9 0 0.0 0 0.0

D 15 3 11 0 0.0 11 100 10 90.9 3 27.3 30 5 17 0 0.0 17 100 5 29.4 0 0.0 60 3 10 0 0.0 10 100 5 50.0 0 0.0

As the spermatozoon penetrates farther into the jelly, the rest of the head plus the neckpiece and tail enter the 54 layer (fig. 5). In general, the heads of most spermatozoa are di- rected toward the egg surface and most of the sperm tends to be rectilinear. At this time, the combined thickness of all jelly layers is about 325-400 pM, thus a spermatozoon 600 p M long begins to enter the 52 layer before the end of its tail has left the 55 surface. Observa- tions on both living and fixed preparations show that spermatozoa usually penetrate directly through the jelly coats without being noticeably deflected a t any layer, but distor- tions occur during hydration or fixation (figs. 6, 7 ) . Upon emerging from the unhydrated jelly layers, i t seems likely that the usual situation is for the sperm head to encounter and penetrate the vitelline envelope almost immediately. In eggs never immersed in water, the capsular chamber is not large and the innermost jelly layer remains fairly close to the egg surface. Some spermatozoa, however, do not enter the egg cytoplasm immediately, but emerge completely from the jelly layers into the capsular chamber (figs. 8-10). In the capsular chamber the living sperm mostly return to the hoop shape characteristic of sperm in the vas deferens (fig. 9). They con- tinue to move, but seem to be incapable of penetrating the vitelline envelope after the first few minutes, thus a sizeable accumulation of sperm is sometimes seen; they usually stop movement about 20 minutes after the beginning of hydration. Also, a t this time the 53 layer has changed its nature and seems to be no longer penetrable by the sperm. Sper- matozoa accumulate a t the 53-54 interface, sometimes becoming contorted (figs. 10, 11, 17); they are occasionally trapped in the

metamorphosing 53 layer or seem to be turned back toward the surface (figs. 10, 18).

Penetration of the vitelline envelope appears to occur at something less than a right angle. In only a few cases has sperm penetration been observed in the living state, but all such penetrations have resembled the situation shown in figure 12. Observations on fixed material are similar (figs. 13, 14). Figure 13 shows a sperm which has only partially penetrated the egg itself. In later stages, the whole head was sometimes observed within the egg just underneath and parallel to the vitelline envelope (fig. 14); in other instances, the head was oriented more directly toward the center of the egg. There were no detectable changes in the egg surface or cytoplasm a t the point of sperm entry. In eggs fixed three to ten minutes after insemination, spermatozoa were observed deeper in the cytoplasm (fig. 15); although the head and neckpiece were readily discernible, the tail could not be dis- tinguished with certainty.

Jelly-covered eggs immersed in water before insemination

In these experiments, eggs from regions D and E of the oviduct were immersed in water for 15, 30 or 60 minutes prior to insemination to allow hydration of the jelly layers. After insemination, eggs were fixed either 15 minutes or 15 hours later. Results are summarized in table 3.

It was noted in serial sections that in all but three region E eggs, the spermatozoa re- mained in or on the 55 layer and showed no evidence of movement into the 54 layer (fig. 20). In the three exceptions, only a few spermatozoa were seen in the 54 jelly and none were observed deeper than the 53 layer. The


55 layer quickly loses its stickiness upon hydration and living spermatozoa do not adhere as well; they tend to move over the jelly without penetrating.

Cleavage was observed in a few of the region D eggs immersed in water for 15 minutes prior to insemination, but no eggs immersed for 30 or 60 minutes showed any cytological evidence of fertilization. Unlike eggs from region E, all region D eggs, regardless of immersion time, had spermatozoa within the jelly layers and in many cases spermatozoa were found in the capsular chamber. Although spermatozoa readily penetrated the 54 jelly of 30 and 60 minute region E eggs, only a few were found beyond the 53 layer. Some spermatozoa had reached the 53-54 boundary and were observed lying parallel to it (fig. 16). Other spermatozoa were looped upon themselves to form small coils embedded in the 54 layer (fig. 17). Still others appeared to have been deflected from their path toward the egg by the 53 layer and many were found with their heads di- rected back towards the 54 periphery (fig. 18). These observations are similar to those made on living material (vide supra), which also show the development of a barrier to sperm penetration in the 53 layer as hydration begins (figs. 10, 11).

In several region E eggs which had been immersed for 30 or more minutes, it was noted in serial sections that a portion of the 55 jelly coat had been fortuitously stripped away, thus exposing the 54 layer. By chance, some spermatozoa were smeared on the jelly coat a t these sites during insemination. It was observed that spermatozoa located on the intact 55 surface of these eggs showed no evidence of movement through the 55 layer. However, those spermatozoa in direct contact with the 54 jelly had penetrated to the 53-54 boundary (fig. 19). The data for these eggs were not included in table 3.

Results from these experiments show that after immersion for 15 minutes or more a change occurs in the 55 layer which makes it impenetrable to spermatozoa. Beyond 15 minutes an alteration of the 53 layer also occurs and blocks the movement of spermatozoa through it. Spermatozoa placed on the 54 surface, however, can move through this layer regardless of the time of immersion.

DISCUSSION

The results of the present study re-empha- size the importance and complexities of the

jelly envelopes of the newt egg in fertilization. They also indicate that the envelopes un- doubtedly have roles other than the facilita- tion of sperm entry into the egg.

The technique of inseminating eggs taken directly from the lower parts of the oviducts with a concentrated sperm suspension from the vas deferens is clearly the most effective of those tried. Essentially the same method gave similar results with Triton alpestris (Hadorn and Fritz, '50) and is effective with Triton palmatus (G. Fankhauser, personal communication) and Pleurodeles waltlii (Picheral, '77). This approach approximates the situation in the female cloaca where insemination ordinarily occurs, in that spermatozoa from the spermatheca are applied to the egg jelly just minutes before the egg is deposited in water. The method would thus be expected to be more effective than the use of diluted sperm suspensions. Our observations suggest, however, that the natural mechanism of insemination is superior to our a t - tempts to mimic i t , particularly with regard to the percentage of eggs cleaving and the much smaller number of sperm utilized in accom- plishing fertilization in the cloaca. There are many possible reasons for the differences, but one suspects that the spermatozoa housed in the female's spermatheca are in a somewhat different state than those in the vas deferens, perhaps having been modified during the com- plex processes involved in spermatophore deposition, transfer of sperm to the female and storage in the spermatheca. In support of this are the observations of Benson ('651, who states that the spermatozoa of Notophthalmus viridescens are in an inactive state as they are transferred via the spermatophore to the female. Among other factors that could ac- count for the superiority of the natural inseminating mechanism might be the environment within the cloaca itself or the way in which the sperm are released from the sper- mathecal tubules as they open upon the surface of the egg jelly. Dent ('70) has shown that the tubules are expanded through much of their length to diameters of 60-80 km, but that near their orifices they narrow to only a few micrometers, suggesting that exiting spermatozoa might be rectilinear in conformation, rather than hoop-shaped, and be oriented perpendicularly to the 55 surface as they are released. From our observations it would appear that this situation might well facilitate sperm penetration.

80 E. W. MCLAUGHLIN A N D A. A. HUMPHRIES, JR.

The fact tha t spermatozoa from the testis were essentially ineffective suggests t ha t some sort of maturation of t he male gamete occurs after i t leaves the testis and enters the male ducts. Our cytological evidence seems to show tha t sperm from the testis are mostly incapable of penetrating the jelly layers, although they can sometimes be seen in active movement on the J 5 surface.

The relative ineffectiveness of dilute sperm suspensions may be partly the result of changes in the jelly envelopes exposed to a n aqueous medium of low ionic strength, rather than any effect on the spermatozoa themselves. Spermatozoa are motile in these suspensions and appear normal, but cytological examination of uncleaved eggs inseminated with them revealed only a few spermatozoa in the outermost jelly and none within the jelly or in the capsular chamber. This may have been due to loss of adhesive properties of the 55 layer, since our results show tha t hydration of the jelly results in loss of stickiness along with loss of penetrability. Observations from life show tha t the sperm move against the hydrated 55 jelly without adhering or penetrating.

We have confirmed for this urodele the repeated observation tha t the presence of jelly is generally required for fertilization of amphibian eggs. Coelomic oocytes and dejellied region E oocytes were fertilized in very low numbers; but the fertilizability of eggs increased with the addition of more jelly layers. The fertilization process in urodele eggs may require a t least some structural order within the jelly for full efficiency. Of interest in this regard is the tendency for spermatozoa to lose their hoop shape and become straightened as they pass through the jelly, particularly through layer 54. Upon entering the capsular chamber, they often reassume their hoop shape, especially as the chamber expands during hydration. Our observations suggest tha t the rectilinear conformation of the sperm may be important in penetration both through the 53 layer and through the vitelline envelope and plasma membrane, and tha t once this conformation is lost, penetration becomes difficult or impossible. In support of the possible role of the 54 layer in straightening the sperm is the work of one of us (Humphries, '66; and unpublished observations) which suggests t ha t layer 54 tends to be somewhat fibrous, with the fibers often lying roughly perpendicular to the egg surface. This might well serve to orient t he

sperm as i t approaches the egg surface. Re- peated observations such as tha t pictured in figure 11, showing sperm orientation in 54 in the living condition, suggest that this layer does indeed play such a role, although there is obviously no absolute control.

Our results are in agreement with the gen- eralization tha t fertilizability tends to in- crease with the acquisition of additional jelly, except for t he finding tha t eggs with only four layers of jelly from region D tend to be slightly more fertilizable than eggs from region E with five layers. Similar results were reported for this species by Hughes ('56). Since eggs inseminated naturally in the cloaca have all five layers, our results a re somewhat surprising, assuming tha t t he peak of fertilizability should occur when the eggs reach the condition in which they are naturally inseminated. Indeed, work with other species (e.g., Nada- mitsu, '57, with Triturus pyrrhogaster; Elin- son, '71, with Rana pipiens; Brun, '74, with Xenopus laeuis) indicates t ha t acquisition of the full complement of jelly layers is associated with the highest level of fertilizability. I t is perhaps significant tha t none of the other amphibians investigated in this matter has an outermost layer of jelly with characteristics of layer 55 of Notophthalmus. Instead, the outermost layer of jelly of these species seems to resemble the 54 of the eggs studied by us. I t may be tha t the 55 layer of this newt egg is chiefly important as the means by which the female attaches the freshly laid egg to vegetation.

Penetration of region A oocytes by spermatozoa appears to be dependent upon the presence of at least some jelly. Failure of many eggs from regions A, B and C to cleave, even though penetrated by spermatozoa, is probably explained partly by immaturity of the eggs, since the first polar body is usually not extruded until the egg is in the anterior part of the oviduct (Jordan, 1893; Humphries, '56); however, a high degree of polyspermy was often seen in some of our eggs and was probably also a factor. In any case, the abnor- mally high degree of polyspermy in these eggs suggests tha t secretions of the oviduct facili- tated sperm penetration, but t ha t the mecha- nisms necessary for t he prevention of excessive polyspermy were undeveloped.

The speed of penetration of spermatozoa through the five layers of jelly is impressive, and indicates t ha t the jelly constitutes little or no hindrance to Penetration. Jordan (18931,

~


in his pioneering study, considered the jelly to be a barrier to sperm penetration and he sought "long and carefully" for a micropyle. He found none and deduced that it took about one to two hours for the sperni to enter the egg. Fankhauser and Moore ('41) observed spermatozoa in the egg cytoplasm as soon as five minutes after the egg was laid, but the time of insemination was not known due to variability in the time of retention of the egg in the cloaca before laying. The present study shows that, with artificial fertilization, sperm may penetrate the oocyte in a s short a time a s one minute, although it would seem tha t an average time is about three to five minutes. This time agrees well with information about anurans: 3 to 5 minutes for Bufo lentiginosus (King, '01); about 10 minutes for Rana tem- poraria (Katagiri, '61, '63b); 10 to 15 minutes for Rana pipiens (Kemp and Istock, '67) ; 3 to 5 minutes for Bufo arenarum (Barbieri and Schugurensky , '6 7 1.

The eggs of Notophthalmus viridescens are naturally polyspermic, but t he degree of polyspermy is usually not great and high levels of polyspermy are generally associated with abnormal development. In naturally inseminated eggs of Notophthalmus, the number of sperm reported in the egg cytoplasm ranges from 1 to 19, but the usual number seems to be around 5 or 6 (Jordan, 1893; Kaylor, '37; Fankhauser and Moore, '41); this was sub- stantiated by the present study. Kaylor ('37) reported that abnormal development occurred when eggs showed more than 13 sperm penetration marks, but i t appears tha t a higher degree of polyspermy may at least occasionally be tolerated (Fankhauser and Moore, '41). Similar degrees of polyspermy have been found for Triturus pyrrhogaster (Street, '401, for Pleurodeles waltlii (Picheral, '771, and for Triton palmatus (Fankhauser, '32). In work with Triton, Fankhauser ('32) employed artificial insemination, with the result tha t many eggs were penetrated by more than the usual number of spermatozoa. Abnormal cleavage was always observed in eggs having more than ten spermatozoa. Our study showed tha t many eggs from regions A, B and C with excessive polyspermy did not cleave or develop.

In the present investigation, artificial insemination appears to have provided a number of sperm on the 55 surface far in excess of the number provided by the natural inseminating mechanism. Nevertheless, eggs

with four or five jelly layers revealed no excessive degree of polyspermy and normal development ensued in almost every artificially fertilized egg of this type. The apparent lack of excessive polyspermy in region D and E eggs has several possible explanations and is of interest in the general question of control of sperm penetration into the egg. We have observed large accumulations of moving spermatozoa in the capsular chamber five to ten minutes after insemination and prior to hydration; moreover, we have observed repeated contacts of living spermatozoa with the vitelline envelope and failure to penetrate. I t is evident t ha t not all the spermatozoa reach- ing the level of the capsular chamber are able to enter the egg itself. This could be due to some incapacity of the sperm, but another possible explanation is tha t a barrier to sperm penetration develops at the level of the vitelline envelope, as seems to be generally the case for anuran eggs (for example, Wolf, '74; Grey et al., '74). Presence of cortical granules and rapid formation of a fertilization membrane are both regular features of anuran eggs, which are usually monospermic, but cortical granules are apparently lacking in urodele eggs (Wartenberg and Schmidt, '61; Wartenberg, '62; Hope et al., '63; Wischnitzer, '661, which a re usually polyspermic. Forma- tion of a barrier to sperm penetration at the level of the vitelline envelope may occur in both types of eggs, but if so, t he situation suggests t ha t urodele eggs, perhaps because of the absence of cortical granules and a rapid cortical reaction, develop the blockade more slow- ly, thus allowing more sperm to enter the cytoplasm.

Although i t may be tha t changes in the vitelline envelope a re of importance in the prevention of excessive levels of polyspermy, there are clearly changes in jelly layers 53 and 55 which hinder or prevent sperm penetration. Similar observations have been made on anuran eggs (Katagiri, '62, '63a; del Pino, ' 7 3 ) . In both natural insemination and our procedure of artificial insemination, exposure of t he newt egg to water follows within a few minutes, thus one may reasonably conclude tha t hydration changes in the jelly will ordinarily serve as a hindrance to penetration of excessive numbers of spermatozoa as far as the capsular chamber.

Our results have clearly shown, then, the es- sential role of t he unhydrated egg jelly in the rapid fertilization process; also clear, how-

82 E. W. McLAUGHLIN AND A. A. HUMPHRIES. J R

ever, a r e the different characteristics of t he hydrated jelly. Upon hydration, jelly layers become a block to sperm penetration and at tha t time probably acquire new functions associated with protection and maintenance of the dcveloping embryo.

AC'KNOW LEDG MENTS

Supported in par t by a grant (GM09878) I'rom the U. S. Public Health Service.

LITERATURE CITED

Baker. ( ' 1.. 1966 Spermatozoa and spermateleosis in the Saiainandridae with electron microscopy of Drernictylus. .I. Tennessee Acad. Sci.. 41: 2-25.

Harbieri. 1'. D.. and A E. Schugurensky 1967 Cambios estructurales en el ovocito de Bufo arenarum durante Ie fecundacion. Acta Zool. L~lloana, 23: 77-82.

Hrnsoii, I) G.. J r . 1965 Chemical and Morphological Changes in the Cloaca1 Glands of t he Newt, Triturus

ns, as Induced by Hormonal Alterations. Doc- sertation, t he University of Virginia.

Bolognani. I,.. A. M. B. Fantin, R. Lusignani and L. Zonta 1966 Presence of sialopolysaccharide components in egg gelatinous mantle of Rana latastei and Bufo uulgaris. ex^ perientia. 22; 601-602.

H. 1974 Studies on fertilization in Xenopus Biol. Repro., I I ' 513-518.

N . I970 The ultrastructure of t he spermatheca in the red spotted newt. J . Morph.. 232: 397-424.

drl Pino. E. M. 1973 Interactions between gametes and environment in the toad Xenopus laeuis (Daudin) and their relationship to fertilization. cJ. Exp. Zool., 185: 1 2 1 - 1 32.

Elinson, R . P. 1971 Fertilization of partially jellied and jellyless oocytes of the frog Raria p ip i ens . J. Exp. Zool.. 176: 415-428.

.~ 1973 Fertilization of frog body-cavity eggs: Horra p r p i i ~ s eggs and Rnna clarnitans sperm. Biol. Re- prod.. X 362 3ti8.

Fankhausvr. G. 1932 Cytological studies on egg fragments of the salamander Triton. 11. The history of t he supernumerary sperm nuclei in normal fertilization and cleavage of fragments containing the egg nucleus. J. Exp. ZOOI , 62: 185-235

Fankhauser. G., and C. Moore 1941 Cytological and experi- mental studies of polyspermy in the newt, Triturus t'rridescens I . Normal fert i l ization. J . Morph., 68: 347-385.

1968 A study of the jelly envelopes sur- rounding the egg of the amphibian, Xenopus laeuis. Biol. Bull.. 135: 501-513.

Grey, R. D.. D. P. Wolf and J. L. Hedrick 1974 Formation t ructurr of t he fertilization envelope in Xenopus . Devel Biol., 96: 44-61.

Hadorn, E.. and U'. Fritz 1950 Experiinentelles zur Be- fruchtungsphysiologie des Tritoneies. Experientia, 6: 93-98.

Hedrick. .I. I . , A. J Sniith. E. C Yurewicz, G. Oliphant and D. P. Wolf 1974 The incorporation and fate of ('JsS)-sulfate in the ,jelly coat of'Xerzopirs laeuzs eggs. Biol. of Re- prod., 1 I : 534.542.

Hope. J . . A. A . Humphries, Jr. and G. H. Bourne 1963 U1- trastructural studies on developing oocytes of t he salamander Trzturus viridescens 1. The relationship between

Freeman. S. B.

follicle cells and developing oocytes. J. Ultrastr . Res., 9: 302-324.

Hughes, W. N. 1956 An Investigation into the Fer- tilizability of Coelomic and Oviducal Eggs of t he Newt. Triturus uiridescens M. S. Thesis, Emory University

Humphries, A. A,, J r . 1956 A study of meiosis in coelomic and oviducal oocytes of Triturus uiridescens. with particular emphasis on the origin of spontaneous polyploidy and the effects of heat shock on the first meiotic division. J . Morph., 99: 97-136.

1966 Observations on the deposition, structure. and cytochemistry of t he jelly envelopes of the egg of the newt, Triturus uirrdescens. Devel. Biol., 13: 214-230.

1970 Incorporation of "S sulfate into t h e oviducts and egg jelly of t he newt, NotophthaZmus uiridescens. Exptl. Cell Res., 59: 157-161.

Humphries. A. A., Jr . , and W. N. Hughes 1959 A study of the polysaccharide histochemistry of t he oviduct of' t he newt, Trrturus uiridescens. Biol. Bull.. 116: 446-451.

Humphries, A. A,, Jr., S. B. Freeman and W. M. Workman 1968 Sialic acid in egg capsules and oviducts of the newt, Notophthalrnus uiridescens. Biol. Bull., 134: 266-271.

Jordan, E. 0. 1893 The habits and development of t he newt iDiernyctyZus uzridescens). J . Morph., 8: 269-366.

Ka tag r i , C. On the fertilizability of the frog egg. I. J. Fac. Sci., Hokkaido Univ., Ser. VI, Zool., 14: 607-613.

1962 On the fertilizability of t he frog egg. 11. Change of t he jelly envelopes in water. ,Japanese J. 2001.. 13: 365-373.

Fertilizability of t he egg of Hyla arborea japonica with special reference to the change of jelly envelopes. Zool. Mag., 72: 23-28.

1963b On the fertilizability of the frog egg. 111. Removal of egg envelopes from unfertilized egg. J . Fac. Sci., Hokkaido Univ., Ser. VI, Zool., 15: 202-211.

Kaylor, C. T. 1937 Experiments on androgenesis in the newt, Triturus uiridescens. J. Exp. Zool., 76: 375-394.

Kemp, N. E., and N. L. Istock 1967 Cortical changes in growing oocytes and in fertilized or pricked eggs of Hana pipiens. J. Cell Biol., 34: 111-122.

King, H . D. 1901 The maturation and fertilization of t he egg of Eufo lentigznosus. J . Morph., 17: 293-350.

Kingsbury, B. F. 1895 Spermatheca and methods of fertilization in some American newts and salamanders. Proc. Amer. Micro. SOC., 17: 261-304.

Lee, P. A. Studies of frog oviducal jelly secretion. I. Chemical analysis of secretory product. J . Exp. Zool., 166: 99-106.

Martinez, N. R. de, and J. M. Olavarria 1973 The sialic acids of toad oviduct mucoprotein. Biochim. Biophys. Acta, 320: 295-300.

Nadamitsu, S. 1957 Fertilization of coelomic and ovidu~ cal eggs of Trz turus pyrrhogaster (Bole). J. Sci.. Hiroshima Univ.,,Ser. B, Div. 1, 17: 51-53.

Pereda, J . 1970 Etude histochimique de la distribution des s ia lomucines dans l 'oviducte e t les gangues muqueuses des ovocytes de Rana pipiens. Comportement dans l'eau des differentes gangues. J. Embryol. exp. Morph., 24: 1-12,

Picheral, B. 1977 La fecondation chez le triton, Pleu- rodele. I. La traversee des enveloppes de l'oeuf par les spermatozoides. J . Ultrastr . Res., 60: 106-120.

Street , J. C. 1940 Experiments on the organization of the unsegmented egg of Trzturus pyrrhogaster. J. Exp. Zool., 85: 383-408.

Wartenberg, H . 1962 Elektronenmikroskopische und histochemische Studien uber die Oogenese der Amphi- bieneizelle. Zeitschr. Zellforsch., 58: 427-486

1961

1963a

1967


Wartenberg, H., and W. Schmidt 1961 Elektronenmikro- Morphogenesis. Vol. 5. M. Abercrombie and J. Brachet. skopische Untersuchungen der strukturellen Veran- eds. Academic Press, New York. derungen im Rindenbereich des Amphibieneies im Ovar Wolf, D. P. 1974 The cortical granule reaction in living und nach der Befruchtung. Zeitschr. Zellforsch., 54: eggs of the toad, Xenopus laeurs. Devel. Biol., 36: 62-71. 118-146. Yurewicz, E. C., G. Oliphant and J. L. Hedrick 1975 The

Wischnitzer, S. 1966 The ultrastructure of the cyto- macromolecular composition of Xenopus laeuis egg Jelly plasm of the developing amphibian egg. In: Advances in coat. Biochemistry, 14: 3101-3107.

Abbreviations

cc, capsular chamber J l , 52, etc., jelly layers J1, 52, etc.

PLATE 1

EXPLANATlON OF FIGURES

1 Jelly layers of a fixed region E egg. The five layers and a part of the capsular chamber are evident. The egg is out of the field a t the lower edge of the picture. Spermatozoa are numerous in 55; a portion of one can be seen in 54 and 53. Bar represents 50 pm.

An uncleaved region B egg showing numerous sperm asters with chromatin in the cytoplasm. Bar represents 100 pm.

Region D eggs fixed five minutes after insemination; numerous spermatozoa are present in the jelly layers and capsular chamber. Bars represent 100 pm and 50 pm.

2

3 , 4

84

JELLY ENVELOPES AND FERTILIZATION E W McLaughlin and A A Humphnes, J r

PLATE 1

85

PLATE 2

EXPLANATION OF FIGURES

All the eggs in figures 5-8 are from region E and were fixed three to ten minutes after insemination.

5, 6 Head of a spermatozoon in the 54 and 53 jelly layers and the 53 and 52 layers, respectively, of region E eggs. Bars represent 100 pm.

Sperm head emergng from the 52 layer into the capsular chamber. Bar represents 50 pm.

Spermatozoa in jelly and capsular chamber. Bar represents 50 pm

7

8

86

JELLY ENVELOPES AND FEHTILlZATlON E. W. McLauphlin and A. A. Humphnes. Jr

PI.ATE 2

87

JELLY ENVELOPES AND PERTI1,IZATION E W McLaughlin and A A Humphnes. Jr

9

10

11

12

13

14

15

16

PLATE 3


Living spermatozoa in the capsular chamber where they have reassumed their crook shape. Bar represents 100 pm.

Living spermatozoa blocked at the 53-54 interface. Bar represents 100 pm.

Living spermatozoa penetrating the 54 layer a t the top of the picture. Bar represents 50 pm

Living sperm lying almost parallel to the vitelline membrane in position for penetration. The egg fills the lower right part of the picture. Bar represents 50 pm.

All eggs in figures 13-16 are from region E.

Head of a spermatozoon penetrating the vitelline membrane and entering the cytoplasm. Bar represents 50 pm.

Spermatozoon in the egg cytoplasm and lying underneath and parallel to the plasma membrane. Bar represents 10 pm,

Spermatozoon found deeper in the egg cytoplasm three minutes after insemination. Bar represents 10 pm.

A spermatozoon lying parallel to the 53 layer of a region D egg immersed in water for 60 minutes prior to insemination. Apparently the 53 layer has blocked progress of the sperm through the jelly. This egg was fixed 15 hours after insemination. Bar represents 50 pm.

88

JELLY ENVELOPES AND FERTILIZATION E W McLaughlin and A A Humphnes, Jr

PLATIS 4

89

JEI.1.Y ENVELOPES AND FERTILIZAI'ION E W McLauphiin and A A Humphries. J r


90

17, 18 Eggs from region D immersed in water for 60 minutes prior to insemination. Spermatozoa moved through the 54 layer but were stopped from penetrating farther a t the 53-54 interface. Bars represent 50 pm.

A region E egg immersed in water for 30 minutes prior to insemination and having a portion of the 54 jelly exposed at the site of insemination. The area to the left of the arrow is the exposed 54 layer while the area to the right has an intact 55 layer. Spermatozoa placed on the 54 surface have penetrated to the 53 layer, whereas those placed on the 55 layer show no evidence of movement farther into the jelly. Bar represents 100 Fm.

Numerous spermatozoa on the 55 surface of an egg immersed in water 60 minutes prior t o insemination. No sperm were observed in the jelly layers, capsular chamber, or cytoplasm. Bar represents 50 pm.

19

20

PLATE 5

the jelly envelopes and fertilization of eggs of the newt, notophthalmus viridescens

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