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Growth of the preimplantation embryo of the northern fur seal and its correlation with changes in uterine protein
DEVELOPMENTAL
Growth
BIOLOGY
26, 3163’2
(1971)
of the Preimplantation Embryo and Its Correlation with Changes JOSEPH
Department
of Molecular.
C.
DANIEL,
of the Northern Fur Seal in Uterine Protein JR.
Cellular, and Developmental Biology, Boulder. Colorado 80302 Accepted
April
University
of Colomdo,
30, 1971
From samples collected during the period prior to implantation. the growth of the fur seal embryo is described and correlated with changes in the reproductive system of the mother and mitotic activity in the cells of the embryo. The embryo enters a “dormant” stage when it becomes a blastocyst and is not “reactivated” until almost 4 months later. During this time mitosis in the cells of the embryo occurs at a very low (mitotic index = 0.5c c and is extraordinarily long tmitotic duration = about 9 hrl so that it takes 50-60 days for the cells to double their number. Activation occurs 1 to 2 weeks before implantation in November and is characterized by a dramatic increase in cell proliferation and overall expansion. In the mother, the corpus luteum increases in size during this time, presumably reflecting the increase in activity reported by others. The composition of the protein components of the uterine fluids changes both quantitatively and qualitatively with activation of the embryo and may result from the increased production of progesterone from the corpus luteum. The change is not related to overall size ofthe uterus because this does not increase until after implantation. INTRODUCTION
The embryo of the northern fur seal remains dormant in the blastocyst stage for most of 4 months (Enders et al., 1946; Pearson and Enders, 1951; Craig, 1964). Implantation is therefore delayed, resulting in a gestation period that extends over almost one full year. [For recent reviews see Peterson, (1968); Harrison and Koo.yman (19681.1 During this time the blastocyst grows very slowly (see Figs. 1 and 2) exhibiting almost no mitotic activity (Daniel. 1967) or nucleic acid synthesis, but consuming oxygen at relatively high levels and synthesizing small amounts of protein (Gulyas and Daniel, 1967, 1969). In some other species having delayed implantation, the embryos are “activated” fairly abruptly immediately prior to the time of implantation, in that accelerated total growth and new cell proliferation become apparent (e.g.. Baevsky, 19631. Changes in the protein of the uterine secretions also occur coincidental with this activation of the blastocyst (Daniel, 1968; Daniel and Krishnan, 1969); such changes 316
also have been observed in some mammals that do not have delayed implantation (e.g., Daniel, 19701. The intention in the current study was to determine whether the fur seal embryo experiences a distinct period of “activation, ” and if so whether it can be correlated with changes in uterine proteins and/or other changes in the genital system. Some analyses of blastocyst growth rates have been done. METHODS
AND
MATERIALS
To establish the growth curve of the diapausing blastocyst. records of the size of 96 embryos collected from animals killed in the annual fur harvest on the Pribilof Islands, Alaska over the last 5 years were utilized. In addition 16 measurements reported by other investigators were available. Determinations of the weights of uterine horns. diameters of corpora lutea, mitotic index of the cells of the embryos, and concentration of uterine protein were done mainly on a series of three samples of 13-24 animals each, collected, respectively. dur-
DANIEL,
JULY
,JR.
Early
Embpogenesis
AUGUSTSEPTEMBER OCTOBERNCNEMERDECEMBER
FK;. 1. Growth of the fur seal embryo during the first 5 months of pregnancy. Filled qmbols represent diameters of embryos imainly blastocysts); open symbols represent crow-rump length measurements.
The
in the Fur Seal
317
ing late July-early August, late September, and early November (specifically November 6) of 1970. The July-August data were collected on St. Paul Island by the author. and uterine flushings were sent frozen to his laboratory at the University of Colorado for protein determinations. The September and November samples were collected by Dr. Mark Keyes. and the entire genit,al tracts were shipped frozen to the author’s laboratory. where the essential measurements were made. Thus. the embryos in the latter samples were dead and usually collapsed at the time they were recovered. Measurements of their size were, therefore, approximations made from the size of the collapsed cell mass and/or the zona pellucida. Also, the uterine fluid protein might be expected to be somewhat higher in these latter samples because the endometrial cells had been partially ruptured by freezing. However, the protein content of 4 uteri from the *July-August collection, which were frozen and thawed before flushing, was not significantly higher than the fresh-flushed samples. The uterine samples and the embryos
Frc. 2. Fur seal blastocysts collected in August. Diameters of these larger specimen is partially collapsed inside of its zona pellucida.
specimens
were
0.15. 0.2. and 0.32 mm.
318
DEVELOPMENTAL
BIOLOGY
were obtained as follows. That horn of the uterus located on the same side as the new corpus luteum was separated by cuts at the cervix and the uterotubal junction, blotted dry, and weighed. It was then flushed with 10 ml of buffered, physiological saline solution. The flushings were collected in large watch glasses so that they could easilv be searched for the embryo. If the embryo was not found. the horn was cut open down its entire length and its inner surface, was irrigated with a relatively forceful stream of saline solution administered from a hypodermic syringe. This latter procedure was frequentlv necessary to dislodge the blastocyst from the prominent rugae that line the uterus. Then the embryo was measured with an ocular micrometer and removed to a microscope slide, where it was stained with acetic orcein and flattened under a cover slip (Tjio and Whang, 19621. The cells and mitotic figures were counted and a mitotic index was determined. Total protein was determined on a 0.1-ml sample of the flushing by the Lowry procedure (Lowry et al.. 1951) and expressed as milligrams of protein per horn. The remainder of the sample was dialyzed 24 hr against distilled water, lyophilized, and fractionated by filtration on Sephadex G-200. The diameter of the corpus luteum was obtained from cross sections made by slicing the ovary as close to the middle of the ovulation papilla as possible and expressed as the average of the long and short diameter. Corpora lutea collected in the November animals were highly bifurcated and lobulated, as noted by Enders et al. (1946) and Craig (1964). so that the diameter measurements were approximation at b?st. RESULTS
AND
VOLUME
26, 19'il
later in October or early in November. Craig (19641 reported the middle of November as the average implantation date. Therefore, one would expect that a collection of embryos taken between the time when growth is reinitiated and the time they implant would be highly variable. This expectation is borne out by the samples collected on November 6 in which, of the 18 embryos recovered, 6 were already implanted and the developmental stage ranged from small (1 mm in diameter) blastocysts up to fetuses of 20 or more somites. With the growth curve derived in Fig. 1 as a base for comparison, determinations of mitotic indices, concentrations of uterine protein, diameters of corpora lutea. and weights of uterine horns were then plotted for the three major collection periods (plus a few other individual samples where available). These results are shown in Figs. 3-6. As demonstrated by the mitotic index, cell proliferation takes place at a very low rate during the period when the blastocyst is “dormant” (Fig;. 3). On an average, only about 0.5[, of the cells are undergoing
DISCUSSION
Figure 1 shows the growth curve obtained when blastocvst diameter is plotted against the date of collection. It demonstrates that fur seal blastocvsts expand verv little throughout the first 3-3.5 months of pregnancy. but then accelerated growth begins
Frcs. 3-6. Comparison of various reproductive parameters with growth of the embryo in the fur seal prior to implantation. Averages are designated b> open symbols. The heay line is each case represents the same gowth curve as shown in Fig. 1. FIG. 3. Mitotic index.
DANIEL.
FIG. 4. Concentration
JR.
of uterine
Earl\, Embryogenesis in the fir Seal
protein.
. 8 . .
319
ber samples that there is a concentration of points between 1 and 2%, and another concentration just above 4% All t,he embryos from which these later indices were obtained were large and already implanting, while the embryos having indices of 1-2c; were still free blastocysts. It is therefore possible that the embryo of the fur seal accelerates growth in a stepwise fashion, one step wherein its initial mitotic activity is doubled or quadrupled together with free blastocyst, expansion, and then a second step wherein it is at least doubled again coincident with implantation and early organogenesis. Cell counts made on the blastocysts from the late July to early August collection ranged from 120 to 447 per embryo, with an average of 290. The late September samples averaged 586 cells per embryo and ranged from 245 to 937. Thus, it required 50-60 days for the number of cells composing a fur seal “dormant” blastocyst to double. Knowing the mitot.ic index and the doubling time, one can calculate the mitotic duration from the formula of Smit,h and Dendy (19621 which we have used
,/*
FIG.
5. Diameter
of corpus luteum.
mitosis at any one time. This rate increases to about 2.35 by early November, presumably having begun to accelerate late in October. Thus, the fur seal embryo is “activated” to new total growth and new cell production within l-2 weeks prior to implantation. It will be noted in the Novem-
FIG. 6. Weight
of uterine
horn.
320
DEVELOPMENTAL
BIOLOGY
earlier in similar discussions of the growth of blastocysts in rabbits (Daniel, 1962) and ferrets (Daniel, 1970). According to this calculation it would require approximately 8.5-10 hr for these cells to divide (as compared to 30 min for rabbit and 80 min for ferret blastocyst cells). The only other alternative explanations would seem t,o be that (1) only a few select cells reproduce (for which there is no evidence), or (2) mitotis occurs in a cyclic manner, for example, only during daylight hours, the time when all these samples were collected. and not at night. However, at best this would account for only about half of the time, the remainder still being extraordinarily long for mitotic duration. Figure 4 shows that the concentration of uterine protein is initially quite low, but has increased about 16-fold by early November. It seems to have already begun to increase somewhat by late September, but we believe this to be the result of several samples having been contaminated by serum proteins which leaked from t,hese frozen samples when the cervical junction was cut to facilitate flushing. The one es-
VOLUME
26, 1971
pecially high sample (6 mgl obviously contained blood. Figure 7 shows the result of fractionating the uterine fluids by Sephadex gel filtration. The four patterns shown represent samples taken: (a) early in the delay period (July-August) when the mitotic index of the dormant blastocyst was 0.5cC and the total protein was less than 1 mg per horn, (b) later in the delay period (late September) when mitosis was still negligible and the protein low. (c) from the November group that had large blastocysts with a mitotic index of l-2’, and about 5 mg of protein per horn, (d) from an animal having an implant.ing embryo with a mitotic index of 4.3”; and a protein concentration of 11 mg per uterine horn. Two major peaks appear in all the samples, but only the intermediate one (I) with greatest concentration between fractions 60-65 is common to all. The highest molecular weight component (Hl found between fractions 38-48 is not prominent in the samples taken during the delay period, although from the shape of the I peak one concludes that this component (H) probably exists in low concentra-
050 t
040
1
A
C
FIG. 7. Qualitative differences in the protein composition of uterine fluids of the fur seal as demonstrated by Sephadex gel filtration of samples collected in (A) early August, (B) late September, (C and D) early November. The August and September animals had “dormant” blastocysts. The November animals had (Cl unattached “active” blastocysts or (D) implanting embryos. See text for further details.
DANIEL,
JR.
Early
Embpogenesis
tion. Other similar analyses of fur seal uterine fluids show a small but discernible H peak (Daniel and Krishnan. 1969). The lowest, molecular weight components (peak L) found in the samples taken during the delay period are absent or insignificant in the November samples. None of the samples has a distinct protein component in the molecular weight range comparable to blastokinin in the rabbit uterus (Krishnan and Daniel. 1967) and which has been demonstrated in some samples of fluid from mink uteri taken near the time of implantation (Daniel. 1968: Daniel and Krishnan, 1969). However, there is a shoulder in component I at the level of fractions 80-85 which appears in the later samples, and that could possibly repreent a similar component. Obviously, the uterine fluid proteins of the fur seal differ both qualitatively and quantitatively between the time when the embryo is dormant and the time it becomes reactivated to implantation. Diameters of corpora lubea are recorded in Fig. 5. On an average, they continue to increase throughout t,he period studied, but the individual diameters are highly variable, especially in the September and November samples. The average measurements reported by Craig (1964) are also shown to increase for the same period and show close agreement with our measurements. During the delay period the corpora lutea are typically at a low level of activity (Pearson and Enders. 19511, but Craig (1964) concludes, from histological evidence, that in the fur seal there is an active luteal phase that persists for about 1 month after ovulation and, following a regressive phase, a new “active luteal phase intervenes prior to implantation,” accompanied enlargement of uterine by enormous glands. Recent studies of the rabbit (Beier, 1968; Petry et al., 1970; Urzua et al., 1970: Arthur and Daniel, in preparation) show that progesterone is the key hormone for regulating the secretion of specific proteins in the uterine lumen. If the same re-
in the Fur Seal
321
lationship exists in the fur seal, then in view of the increase in size and activity of the corpus luteum, the observed changes in uterine protein (shown in Fig. 4) would be predictable. When fur seal blastocysts are incubated in oitro in medium supplemented with progesterone, no effect on growth is seen (Daniel, 1967). but if the medium contains a specific protein (blastokinin) from the rabbit uterus, mitotic activity is stimulated (Daniel and Krishnan. 1969). Thus, progesterone does not affect the embryo directly but rather would seem to exert its influence by stimulating essential protein secretion in the uterus. Figure 6 records uterine horn weight, for the preimplantation period when the weight of the contained embryo is insignificant. There appears to be no progressive increase and no clear correlation with any of the other parameters studied. When we compared uterine horn weight with protein concentration on an individual sample basis, we were unable to demonstrate any direct correlation: small horns for example can yield as much protein (or as little) as large horns. Thus, the increase in protein concentration seen in the fur seal uterus is not the direct product of an increase in uterine size. Craig (1963), using horn circumference as the criterion, notes that the pregnant uterine horn does not change size significantly in the samples she collected from July through late October, but beginning in December. an increase is noted which of course continues progressively through to parturition in the following June. In the report edited by Hacker (1969). the average diameter of the uterine horns of fur seals collected in Setpember. October, and November remains constant at about 17.5 mm. The author is grateful to Dr. Mark Keyes for help in the collection of the material used in these studies. and to Mr. Bryan Cowan for technical assistance. This work was supported in part by N.S.F. Grant No. GB-6363, N.I.H. Grant No. HD-0248”. and N.I.H. Grant No. HD-01165.
322
DEVELOPMENTAL
BIOLI
REFERENCES
BAEVSKY, U. B. (1963). The effects of embryonic diapause on the nuclei and mitotic activity of mink and rat blastocysts. In “Delayed Implantation” (A. C. Enders, ed.), pp. 141-155. Univ. of Chicago Press, Chicago, Illinois. BEIER, H. M. (1968). Uteroglobin: a hormone sensitive endometrial protein involved in blastocyst development. Biochim. Bioph~s. Acta 160, 289-291. CRAIG, A. M. (1963). Key to the reproductive condition of female fur seals (Callorhinus ursinus) and the reproductive cycle of mature female fur seals. Fish. Res. Bd. l%bl. 25 p. CRAIG, A. M. (1964). Histology of reproduction and the estrus cycle in the female fur seal, Callorhinus ursinu.s. J. Fish. Res. Bd. Can. 21, 773-811. DANIEL, J. C. (1962). Early growth of rabbit trophoblast. Amer. Natur. 98, 85-97. DANIEL, J. C. (1967). Preliminary studies on the diapausing blastocyst of the northern fur seal. Amer. Zool. 7, 757. DANIEL, J. C. (1968). Comparison of electrophoretic patterns of uterine fluids from rabbits and mammals having delayed implantation. Comp. Biochem. Physiol. 24, 297-300. DANIEL, J. C. (1970). Coincidence of embryonic growth and uterine protein in the ferret. J. Embyvol. Exp. Morphol. 24, 305-312. DANIEL, J. C., and KRISHNAN, R. S. ( 1969). Studies on the relationship between uterine fluid components and the diapausing state of blastocysts from mammals having delayed implantation. J. Exp. Zoo/. 172, 267-282. ENDERS, R. K., PEARSON, 0. P., and PEARSON, A. K. (1946). Certain aspects of reproduction in the fur seal. An&. Rec. 94, 213-227. Frscus, C. H., BAINES, G. A., and WILKE, F. (1964). Pelagic fur seal investigations, Alaskan waters 1962. Spec. Sci. Rep. Fisheries, No. 475, 57 pg. GULYAS, B. J., and DANIEL, J. C. (1967). Oxygen consumption in diapausing blastocysts. J. Cell. Physiol. 70, 33-36. GULYAS, B. J., and DANIEL, J. C. (1969). Incorpora-
DGY
VOLUME
26, 1971
tion of labeled nucleic acid and protein precursors by diapausing and nondiapausing blastocysts. Biol. Reprod. 1, 11-20. HACKER, R. L. (1969). Fur seal investigations 1966. Spec. Sci. Rep. Fisheries, No. 584, 123 pg. HARRISON, R. J., and KOOYMAN, G. L. (1968). General physiology of the pinnipedia. In “The Behavior and Physiology of Pinnipeds” (R. J. Harrison, R. C. Hubbard, R. S. Peterson. C. E. Rice, and R. d. Schusterman, eds.), Chap. 7. Appleton, New York. KRISHNAN, R. S., and DANIEL. J. C. (1967). “Blastokinin”-An Inducer and regulator of blastocyst development in the rabbit uterus. Science 158, 490-492. LOWRY. 0. H., ROSEBROKJCH, N. J.. FARR, A. L.. and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. PEARSON, A. K., and ENDERS, R. K. (1951). Further observations on the reproduction of the Alaskan fur seal. Anut. Rec. 111, 695-712. PETERSON, R. S. (1968). Social behavior in pinnipeds with particular reference to the northern fur seal. In “The Behavior and Physiology of Pinnipeds.” (R. J. Harrison, R. C. Hubbard, R. S. Peterson. C. E. Rice, and R. J. Schusterman, eds.), Chap. 1 Appleton, New York. PETRY, G., KOHNEL, W., and BEIER, H. M. (1970). Untersuchungen zur hormonellen Regulation der Praeimplantationsphase der GraviditB’t. I. Histologische, topochemische und biochemische Analvsen am normalen Kaninchenuterus. CFtobiologie 2, l-32. SMITH, C. L.. and DENDY, P. P. (1962). Relation between mitotic index, duration mitosis, generation time and fraction of dividing cells in a cell population. Nature (London) 193, 555-556. TJIO, J. H., and WHANG, J. (1962). Chromosome preparations of bone marrow cells without prior in oitro culture or in uiuo colchicine administration. Stain Technol. 37, 17-20. URZUA, M. A., STRAMBAUGH, R., FLICKINGER, G., and MASTRIOANNI, L. (1970). Uterine and oviduct fluid protein patterns in the rabbit before and after ovulation. Fert. Steril. 21, 860-865.
Growth
BIOLOGY
26, 3163’2
(1971)
of the Preimplantation Embryo and Its Correlation with Changes JOSEPH
Department
of Molecular.
C.
DANIEL,
of the Northern Fur Seal in Uterine Protein JR.
Cellular, and Developmental Biology, Boulder. Colorado 80302 Accepted
April
University
of Colomdo,
30, 1971
From samples collected during the period prior to implantation. the growth of the fur seal embryo is described and correlated with changes in the reproductive system of the mother and mitotic activity in the cells of the embryo. The embryo enters a “dormant” stage when it becomes a blastocyst and is not “reactivated” until almost 4 months later. During this time mitosis in the cells of the embryo occurs at a very low (mitotic index = 0.5c c and is extraordinarily long tmitotic duration = about 9 hrl so that it takes 50-60 days for the cells to double their number. Activation occurs 1 to 2 weeks before implantation in November and is characterized by a dramatic increase in cell proliferation and overall expansion. In the mother, the corpus luteum increases in size during this time, presumably reflecting the increase in activity reported by others. The composition of the protein components of the uterine fluids changes both quantitatively and qualitatively with activation of the embryo and may result from the increased production of progesterone from the corpus luteum. The change is not related to overall size ofthe uterus because this does not increase until after implantation. INTRODUCTION
The embryo of the northern fur seal remains dormant in the blastocyst stage for most of 4 months (Enders et al., 1946; Pearson and Enders, 1951; Craig, 1964). Implantation is therefore delayed, resulting in a gestation period that extends over almost one full year. [For recent reviews see Peterson, (1968); Harrison and Koo.yman (19681.1 During this time the blastocyst grows very slowly (see Figs. 1 and 2) exhibiting almost no mitotic activity (Daniel. 1967) or nucleic acid synthesis, but consuming oxygen at relatively high levels and synthesizing small amounts of protein (Gulyas and Daniel, 1967, 1969). In some other species having delayed implantation, the embryos are “activated” fairly abruptly immediately prior to the time of implantation, in that accelerated total growth and new cell proliferation become apparent (e.g.. Baevsky, 19631. Changes in the protein of the uterine secretions also occur coincidental with this activation of the blastocyst (Daniel, 1968; Daniel and Krishnan, 1969); such changes 316
also have been observed in some mammals that do not have delayed implantation (e.g., Daniel, 19701. The intention in the current study was to determine whether the fur seal embryo experiences a distinct period of “activation, ” and if so whether it can be correlated with changes in uterine proteins and/or other changes in the genital system. Some analyses of blastocyst growth rates have been done. METHODS
AND
MATERIALS
To establish the growth curve of the diapausing blastocyst. records of the size of 96 embryos collected from animals killed in the annual fur harvest on the Pribilof Islands, Alaska over the last 5 years were utilized. In addition 16 measurements reported by other investigators were available. Determinations of the weights of uterine horns. diameters of corpora lutea, mitotic index of the cells of the embryos, and concentration of uterine protein were done mainly on a series of three samples of 13-24 animals each, collected, respectively. dur-
DANIEL,
JULY
,JR.
Early
Embpogenesis
AUGUSTSEPTEMBER OCTOBERNCNEMERDECEMBER
FK;. 1. Growth of the fur seal embryo during the first 5 months of pregnancy. Filled qmbols represent diameters of embryos imainly blastocysts); open symbols represent crow-rump length measurements.
The
in the Fur Seal
317
ing late July-early August, late September, and early November (specifically November 6) of 1970. The July-August data were collected on St. Paul Island by the author. and uterine flushings were sent frozen to his laboratory at the University of Colorado for protein determinations. The September and November samples were collected by Dr. Mark Keyes. and the entire genit,al tracts were shipped frozen to the author’s laboratory. where the essential measurements were made. Thus. the embryos in the latter samples were dead and usually collapsed at the time they were recovered. Measurements of their size were, therefore, approximations made from the size of the collapsed cell mass and/or the zona pellucida. Also, the uterine fluid protein might be expected to be somewhat higher in these latter samples because the endometrial cells had been partially ruptured by freezing. However, the protein content of 4 uteri from the *July-August collection, which were frozen and thawed before flushing, was not significantly higher than the fresh-flushed samples. The uterine samples and the embryos
Frc. 2. Fur seal blastocysts collected in August. Diameters of these larger specimen is partially collapsed inside of its zona pellucida.
specimens
were
0.15. 0.2. and 0.32 mm.
318
DEVELOPMENTAL
BIOLOGY
were obtained as follows. That horn of the uterus located on the same side as the new corpus luteum was separated by cuts at the cervix and the uterotubal junction, blotted dry, and weighed. It was then flushed with 10 ml of buffered, physiological saline solution. The flushings were collected in large watch glasses so that they could easilv be searched for the embryo. If the embryo was not found. the horn was cut open down its entire length and its inner surface, was irrigated with a relatively forceful stream of saline solution administered from a hypodermic syringe. This latter procedure was frequentlv necessary to dislodge the blastocyst from the prominent rugae that line the uterus. Then the embryo was measured with an ocular micrometer and removed to a microscope slide, where it was stained with acetic orcein and flattened under a cover slip (Tjio and Whang, 19621. The cells and mitotic figures were counted and a mitotic index was determined. Total protein was determined on a 0.1-ml sample of the flushing by the Lowry procedure (Lowry et al.. 1951) and expressed as milligrams of protein per horn. The remainder of the sample was dialyzed 24 hr against distilled water, lyophilized, and fractionated by filtration on Sephadex G-200. The diameter of the corpus luteum was obtained from cross sections made by slicing the ovary as close to the middle of the ovulation papilla as possible and expressed as the average of the long and short diameter. Corpora lutea collected in the November animals were highly bifurcated and lobulated, as noted by Enders et al. (1946) and Craig (1964). so that the diameter measurements were approximation at b?st. RESULTS
AND
VOLUME
26, 19'il
later in October or early in November. Craig (19641 reported the middle of November as the average implantation date. Therefore, one would expect that a collection of embryos taken between the time when growth is reinitiated and the time they implant would be highly variable. This expectation is borne out by the samples collected on November 6 in which, of the 18 embryos recovered, 6 were already implanted and the developmental stage ranged from small (1 mm in diameter) blastocysts up to fetuses of 20 or more somites. With the growth curve derived in Fig. 1 as a base for comparison, determinations of mitotic indices, concentrations of uterine protein, diameters of corpora lutea. and weights of uterine horns were then plotted for the three major collection periods (plus a few other individual samples where available). These results are shown in Figs. 3-6. As demonstrated by the mitotic index, cell proliferation takes place at a very low rate during the period when the blastocyst is “dormant” (Fig;. 3). On an average, only about 0.5[, of the cells are undergoing
DISCUSSION
Figure 1 shows the growth curve obtained when blastocvst diameter is plotted against the date of collection. It demonstrates that fur seal blastocvsts expand verv little throughout the first 3-3.5 months of pregnancy. but then accelerated growth begins
Frcs. 3-6. Comparison of various reproductive parameters with growth of the embryo in the fur seal prior to implantation. Averages are designated b> open symbols. The heay line is each case represents the same gowth curve as shown in Fig. 1. FIG. 3. Mitotic index.
DANIEL.
FIG. 4. Concentration
JR.
of uterine
Earl\, Embryogenesis in the fir Seal
protein.
. 8 . .
319
ber samples that there is a concentration of points between 1 and 2%, and another concentration just above 4% All t,he embryos from which these later indices were obtained were large and already implanting, while the embryos having indices of 1-2c; were still free blastocysts. It is therefore possible that the embryo of the fur seal accelerates growth in a stepwise fashion, one step wherein its initial mitotic activity is doubled or quadrupled together with free blastocyst, expansion, and then a second step wherein it is at least doubled again coincident with implantation and early organogenesis. Cell counts made on the blastocysts from the late July to early August collection ranged from 120 to 447 per embryo, with an average of 290. The late September samples averaged 586 cells per embryo and ranged from 245 to 937. Thus, it required 50-60 days for the number of cells composing a fur seal “dormant” blastocyst to double. Knowing the mitot.ic index and the doubling time, one can calculate the mitotic duration from the formula of Smit,h and Dendy (19621 which we have used
,/*
FIG.
5. Diameter
of corpus luteum.
mitosis at any one time. This rate increases to about 2.35 by early November, presumably having begun to accelerate late in October. Thus, the fur seal embryo is “activated” to new total growth and new cell production within l-2 weeks prior to implantation. It will be noted in the Novem-
FIG. 6. Weight
of uterine
horn.
320
DEVELOPMENTAL
BIOLOGY
earlier in similar discussions of the growth of blastocysts in rabbits (Daniel, 1962) and ferrets (Daniel, 1970). According to this calculation it would require approximately 8.5-10 hr for these cells to divide (as compared to 30 min for rabbit and 80 min for ferret blastocyst cells). The only other alternative explanations would seem t,o be that (1) only a few select cells reproduce (for which there is no evidence), or (2) mitotis occurs in a cyclic manner, for example, only during daylight hours, the time when all these samples were collected. and not at night. However, at best this would account for only about half of the time, the remainder still being extraordinarily long for mitotic duration. Figure 4 shows that the concentration of uterine protein is initially quite low, but has increased about 16-fold by early November. It seems to have already begun to increase somewhat by late September, but we believe this to be the result of several samples having been contaminated by serum proteins which leaked from t,hese frozen samples when the cervical junction was cut to facilitate flushing. The one es-
VOLUME
26, 1971
pecially high sample (6 mgl obviously contained blood. Figure 7 shows the result of fractionating the uterine fluids by Sephadex gel filtration. The four patterns shown represent samples taken: (a) early in the delay period (July-August) when the mitotic index of the dormant blastocyst was 0.5cC and the total protein was less than 1 mg per horn, (b) later in the delay period (late September) when mitosis was still negligible and the protein low. (c) from the November group that had large blastocysts with a mitotic index of l-2’, and about 5 mg of protein per horn, (d) from an animal having an implant.ing embryo with a mitotic index of 4.3”; and a protein concentration of 11 mg per uterine horn. Two major peaks appear in all the samples, but only the intermediate one (I) with greatest concentration between fractions 60-65 is common to all. The highest molecular weight component (Hl found between fractions 38-48 is not prominent in the samples taken during the delay period, although from the shape of the I peak one concludes that this component (H) probably exists in low concentra-
050 t
040
1
A
C
FIG. 7. Qualitative differences in the protein composition of uterine fluids of the fur seal as demonstrated by Sephadex gel filtration of samples collected in (A) early August, (B) late September, (C and D) early November. The August and September animals had “dormant” blastocysts. The November animals had (Cl unattached “active” blastocysts or (D) implanting embryos. See text for further details.
DANIEL,
JR.
Early
Embpogenesis
tion. Other similar analyses of fur seal uterine fluids show a small but discernible H peak (Daniel and Krishnan. 1969). The lowest, molecular weight components (peak L) found in the samples taken during the delay period are absent or insignificant in the November samples. None of the samples has a distinct protein component in the molecular weight range comparable to blastokinin in the rabbit uterus (Krishnan and Daniel. 1967) and which has been demonstrated in some samples of fluid from mink uteri taken near the time of implantation (Daniel. 1968: Daniel and Krishnan, 1969). However, there is a shoulder in component I at the level of fractions 80-85 which appears in the later samples, and that could possibly repreent a similar component. Obviously, the uterine fluid proteins of the fur seal differ both qualitatively and quantitatively between the time when the embryo is dormant and the time it becomes reactivated to implantation. Diameters of corpora lubea are recorded in Fig. 5. On an average, they continue to increase throughout t,he period studied, but the individual diameters are highly variable, especially in the September and November samples. The average measurements reported by Craig (1964) are also shown to increase for the same period and show close agreement with our measurements. During the delay period the corpora lutea are typically at a low level of activity (Pearson and Enders. 19511, but Craig (1964) concludes, from histological evidence, that in the fur seal there is an active luteal phase that persists for about 1 month after ovulation and, following a regressive phase, a new “active luteal phase intervenes prior to implantation,” accompanied enlargement of uterine by enormous glands. Recent studies of the rabbit (Beier, 1968; Petry et al., 1970; Urzua et al., 1970: Arthur and Daniel, in preparation) show that progesterone is the key hormone for regulating the secretion of specific proteins in the uterine lumen. If the same re-
in the Fur Seal
321
lationship exists in the fur seal, then in view of the increase in size and activity of the corpus luteum, the observed changes in uterine protein (shown in Fig. 4) would be predictable. When fur seal blastocysts are incubated in oitro in medium supplemented with progesterone, no effect on growth is seen (Daniel, 1967). but if the medium contains a specific protein (blastokinin) from the rabbit uterus, mitotic activity is stimulated (Daniel and Krishnan. 1969). Thus, progesterone does not affect the embryo directly but rather would seem to exert its influence by stimulating essential protein secretion in the uterus. Figure 6 records uterine horn weight, for the preimplantation period when the weight of the contained embryo is insignificant. There appears to be no progressive increase and no clear correlation with any of the other parameters studied. When we compared uterine horn weight with protein concentration on an individual sample basis, we were unable to demonstrate any direct correlation: small horns for example can yield as much protein (or as little) as large horns. Thus, the increase in protein concentration seen in the fur seal uterus is not the direct product of an increase in uterine size. Craig (1963), using horn circumference as the criterion, notes that the pregnant uterine horn does not change size significantly in the samples she collected from July through late October, but beginning in December. an increase is noted which of course continues progressively through to parturition in the following June. In the report edited by Hacker (1969). the average diameter of the uterine horns of fur seals collected in Setpember. October, and November remains constant at about 17.5 mm. The author is grateful to Dr. Mark Keyes for help in the collection of the material used in these studies. and to Mr. Bryan Cowan for technical assistance. This work was supported in part by N.S.F. Grant No. GB-6363, N.I.H. Grant No. HD-0248”. and N.I.H. Grant No. HD-01165.
322
DEVELOPMENTAL
BIOLI
REFERENCES
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