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Ultrastructure of fetal liver at term and during prolonged gestation in the rat
Ultrastructure of fetal liver at term and during prolonged gestation in the rat JAMES A. THLIVERIS, PH.D. Winnipeg, Manitoba .• Canada The ultrastructure of the fetal rat liver is described at term (Day 22) and during prolonged gestation (Days 23, 24, and 25). Pregnancies were prolonged by daily subcutaneous injections of progesterone to the mothers from Days 20 through 24 of gestation. At term, fetal rat hepatocytes showed a large glycogen-rich area and one free of glycogen, containing cytoplasmic organelles. In contrast, hepatocytes during prolonged gestation (Days 24 and 25) were characterized by depletion of glycogen, marked proliferation of smooth endoplasmic reticulum, and an increase in the numbers of microbodies. Moreover, the smooth endoplasmic reticulum, which is often seen in communication with rough endoplasmic reticulum, extends into the glycogen areas. This latter observation is of particular interest in view of the fact that smooth endoplasmic reticulum has been implicated in glycogenolysis. In addition to the morphologic findings, meconium, which is indicative of fetal distress, was also observed in the gestational sacs on Days 24 and 25.
ious fetal organs. The first report in the series revealed ultrastructural changes in the fetal adrenal gland which were interpreted as an indication of increased activity in response to fetal distress. 20 The aim of the current study was to determine whether ultrastructural changes occurred in the fetal liver during prolonged pregnancy.
F E T A L A N D N E 0 N A T A L distress as a consequence of prolonged pregnancy long has been the subject of numerous studies. Previous investigations have focused mainly on the problems of placental insufficiency/· ~. 4 • H. 1n the postmaturity syndrome/· 4 • "'· 1 '· 1 ~· 31 and hypoglycemia in the neonate. 1 1. 22 On the other hand, little is known concerning the effects of prolonged pregnancy on the internal organs of the fetus. The present report is part of a series of electron microscopic investigations, with the rat used as an experimental model, to study the effects of prolonged pregnancy on var-
Material and methods
Twenty adult female Sprague-Dawley rats weighing 180 to 220 grams were used for this study. While in the proestrous phase, they were mated with male rats for 2 hours. The first day of pregnancy was designated as commencing 24 hours after spermatozoa were found in vaginal smears. Accordingly, the female rats deliver normally on Day 22 of pregnancy. The tissues examined consisted of livers from fetuses taken from 12 female rats whose gestational periods were prolonged 1, 2, or 3 days beyond term (Day 22). Prolonged pregnancy was achieved by daily subcutaneous injections of 7 mg. of progesterone*
From the Department of Anatomy, Faculty of Medicine, University of Manitoba. Supported by the Medical Research Council of Canada, Grant No. MA4740. Received for publication july 6, 1973. Revised September 6, 1973. Accepted September 17, 1973. Reprint requests: Dr. james A. Thliveris, Department of Anatomy, The University of Manitoba, 750 Bannatyne Ave., Winnipeg, Manitoba R3E OW3, Canada.
*Proluton, Schering Corp., lOll New Jerspy 07083.
864
Morri~
Aw., Vnion,
Ultrastructure of fetal liver
Volume 118 Number 6
in sesame oil from the twentieth through the twenty-fourth days of gestation. To evaluate any conceivable effect exogenous progesterone might have on fetal liver morphology, 4 pregnant female rats were given 7 mg. of progesterone daily from Day 14 through Day 21 of gestation. Fetal livers from this group were compared with livers of fetuses taken from normal term pregnancies. Laparotomy was performed on pregnant female rats under pentobarbital* anesthesia (5 mg. per 100 Gm. of body weight) on Days 22, 23, 24, and 25 (8 animals on Day 22 and 4 on each of the remaining 3 days) . Each fetal liver was removed after laparotomy, sectioned, and fixed for 2 hours at 4° C. in 3 per cent glutaraldehyde in O.lM phosphate buffer (pH 7.4). All tissues were rinsed for 24 hours at 4° C. in O.lM phosphate buffer (pH 7.4) containing 0.2M sucrose. Tissues were then postfixed for 2 hours at 4° C. in 2 per cent osmium tetroxide in O.lM phosphate buffer (pH 7.4). Following rapid dehydration in ascending concentrations of ethanol, the tissues were embedded in Epon 812. 13 Thick (1 p,) sections were stained with toluidine blue and examined for routine orientation. Thin sections were stained with uranyl acetate and lead citrate, viewed, and photographed in an electron microscope. t Results
Term (Day 22). At term, fetal rat hepatocytes were characterized by two discrete areas, one filled largely by glycogen and a second occupied by cytoplasmic organelles (Fig. 1). Glycogen was present mainly in the particulate or beta form, 15 but the larger rosettes or alpha particulates15 were also found (Fig. 2). Mitochondria varied in size and shape and contained lamellar cristae. The Golgi membranes and associated vesicles were well developed. Lysosomal-like
*Nembutal, Abbott Laboratories, 14th and Sheridan Rd., North Chicago, Illinois 60064. tEM 300, Philips Electronic Instruments, 750 S. Fulton Ave., Mount Vernon, New York 10550.
865
structures and microbodies were infrequently observed (Fig. 2) . The endoplasmic reticulum was predominately of the rough-surfaced type and was usually seen as parallel stacks of tubules, which frequently showed dilatations. Very few profiles of smooth reticulum were seen. Lipid droplets were also present. Prolonged gestation (Days 23, 24, and 25). The ultrastructure of fetal hepatocytes on Day 23 of gestation was similar to that described at term. On Days 24 and 25, several striking changes were present. Marked depletion of glycogen (Fig. 3) as well as the appearance of large quantities of smooth-surfaced endoplasmic reticulum was observed (Fig. 4). The smooth endoplasmic reticulum was always seen in association with the glycogen areas (Fig. 4). Communications between rough- and smoothsurfaced reticulum were frequently present (Fig. 5). The rough-surfaced endoplasmic reticulum did not appear altered from that seen at term. In addition, microbodies appeared more numerous on Days 24 and 25 than at term. Other organelles such as nuclei, Golgi complexes, and lysosomal-like structures did not appear altered. Greater numbers of hepatocytes exhibiting the above changes were seen on Day 25 rather than on Day 24. In contrast, while cells similar to those seen at term were detected on both days, fewer were observed on Day 25 than on Day 24. Fetal hepatocytes of preterm progesterone-treated female rats. The morphology of hepatocytes from fetuses of preterm progesterone-treated female rats was similar to that described for the term fetal hepatocytes. This observation ruled out an effect by progesterone on fetal hepatocyte morphology. In addition to the ultrastructural findings, fetal meconium staining, which is indicative of fetal distress, 4 was observed on Days 24 and 25 but not at term or on Day 23. Comment
The results of the present investigation have shown that glycogen was markedly de-
866
Thliveris Am .
1 1a rch 15, 197' Gy necol
J. O hst c·t.
Fig. 1. Fetal hepatocytes at term (Day 22 ) . These cells ·are characterized by glycogen-rich a reas ( Gl) and glycogen-free areas containing numerous cytoplasmic organelles. L , Lipid . ( Origina l magnification x7,400. )
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Ultrastructure of fetal liver
Fig. 2. Portion of a fetal hepatocyte at term (Day 22). Glycogen (GI) is present mainly in the particulate or beta form ( ~) . Larger rosettes or alpha particles are also seen (-I-+ ). Rough endoplasmic reticulum (RER) is abundant while smooth endoplasmic reticulum ( SER) is scarce. Mitochondria (M), Golgi apparatuses (G ), and microbodies (Mb) are shown. (Original magnification x19, 700.)
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Fig. 3. Fetal hepatocytes on Day 24 of gestation. Hepatocytes at this stage show marked depletion of glycogen (Gl) and abundant smooth endoplasmic reticulum (SER). Increased numbers of microbodies ( Mb) are also seen. (Original magnification x7 ,400.)
Volume 118 Number 6
Ultrastructure of fetal liver
Fig. 4. Portion of a fetal hepatocyte on Day 24 of gestation. Numerous profiles of smooth endoplasmic reticulum (SER) are seen associated with glycogen (Gl) areas. Mitochondria (M) and rough endoplasmic reticulum (RER) are shown. (Original magnification x32,600.)
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Marclt 15, l9i4 Am. j. Obstet. Gynecol.
Fig. 5. Portion of a fetal hepatocyte showing communication between elements of the rough and smooth endoplasmic reticulum (arrows). Microbodies ( M) are shown. (Original magnification x26, 700.)
pleted from fetal hepatocytes during prolonged gestation (Days 24 and 25 ) . Concomitant with glycogen depletion was the appearance of large quantities of smooth endoplasmic reticulum which has been shown to be derived from rough endoplasmic reticulum.1~ Similar ultrastructural findings have been reported previously in hepatocytes of newborn rats"· n as well as in fasting adult rats.-' Moreover, associated with smooth endoplasmic reticulum is the enzyme glucose6-phosphatase which quantitatively increases during glycogenolysis. 1 " One might conclude that an inverse relationship existed between glycogen and smooth endoplasmic reticulum and glycogen and glucose-6-phosphatase as well as a direct relationship between smooth endoplasmic reticulum and the enzyme. However, Dawkins 7 has shown that during prolonged gestation in rabbits glucose-6-phosphatase did not increase but remained at the relatively low values present prior to birth. It is also known that the hepatic glycogen content in this species decreases substantially during prolonged pregnancy. 1n A marked increase in the enzyme level did occur 24 hours after delivery, the values being similar to those in 24-hour-old
rabbits delivered at term. Dawkins" has also provided evidence in rats that, while large amounts of hepatic glycogen were mobilized during the first 12 hours of life, the depletion was not dependent upon the magnitude of the rise in glucose-6-phosphatase. Ry utilization of inhibitors of protein synthesis, the normal high increase in enzyme activity was obstructed but not entirely prevented. Moreover, glycogen in addition to glucose-6phosphate levels in this condition decreased as readily as in untreated newborn animals. It would appear from the present investigation that while gJucose-6-phosphatase activity most likely did not increase during prolonged pregnancy the quantity present apparently was sufficient to allow glycogen mobilization to occur. The function of microbodies in hepatocytes remains unsettled, and the significance of the increased numbers of these structures observed in the present study is unknown. In prolonged pregnancy in man, fetal weight loss"· ' · 10 and an increased tendency toward neonatal hypoglycemia n. 12 occur iu those infants showing the postmaturity syndrome. Both of these conditions have been attrib11ted to placental insufficiency 1t is
Volume 118 Number 6
conceivable that if maternal nutrients were no longer adequately transferred by the placenta glycogen mobilization in the fetal liver possibly could sustain the fetus. The hypoglycemia present following delivery of a newborn infant in a prolonged gestation
Ultrastructure of fetal liver
871
might be partly because of a depletion of the large glycogen reserve, which under normal conditions at birth is readily mobilized in an attempt to maintain blood glucose levels until feeding comrnencesY
REFERENCES
1. Bleischer, N. A., Brown, J. B., and Townsend, L.: AM. J. OasTET. GYNECOL. 103: 496, 1969. 2. Browne, J. C. M.: AM. ]. 0BSTET. GYNECOL. 85: 573, !963. 3. Cardell, R. R.: Am. J. Anat. 131: 21, 1971. 4. Clifford, S. H.: J. Pediatr. 44: 1, 1954. 5. Dallner, G., Siekevitz, P., and Palade, G. E.: J. Cell Bioi. 30: 73, 1966. 6. Dallner, G., Siekevitz, P., and Palade, G. E.: J. Cell Bioi. 30: 9 7, 1966. 7. Dawkins, M. R. J.: Nature (Land.) 191: 72, 1961. 8. Dawkins, M. R. J.: Ann. N. Y. Acad. Sci. 111: 203, 1963. 9. Fischer-Rasmussen, W., Aegidius, J.: Acta Obstet. Gynecol. Scand. 51: 25, 1972. 10. Gruenwald, P.: AM. J. OaSTET. GYNECOL. 98: 503, 1964. 11. Khattab, A. K., and Forfur, J. 0.: Bioi. Neonat. 18: 1, 1971.
12. Leskes, A., Siekevitz, P., and Palade, G. E.: J. Cell Bioi. 49: 264, 1971. 13. Luft, J. H.: J. Biophys. Biochern. Cytol. 9: 409, 1961. 14. McKiddie, J. M.: Obstet. Gynecol. Survey 5: 44, 1950. 15. Revel, J. P.: J. Histochern. Cytochern. 12: !04, 1964. 16. Roux, J. F., Romrney, S. L., and Dinnerstein, A.: AM. J. OaSTET. GYNECOL. 90: 546, 1964. 17. Shelley, H. J.: Br. Med. Bull. 17: 137, 1961. 18. Sjostedt, S., Englesson, G., and Roath, G.: Arch. Dis. Child. 33: 123, 1958. 19. Strand, A.: Acta Obstet. Gynecol. Scand. (Suppl.) 45: 127, 1966. 20. Thliveris, J. A., and Connell, R. S.; Anat. Rec. 175: 607, 1973. · 21. Walker, J.: J. Obstet. Gynaecol. Brit. Ernp. 61: 162, 1954. 22. Zuspan, F. P.: J. Reprod. Med. 4: 77, 1970.
ious fetal organs. The first report in the series revealed ultrastructural changes in the fetal adrenal gland which were interpreted as an indication of increased activity in response to fetal distress. 20 The aim of the current study was to determine whether ultrastructural changes occurred in the fetal liver during prolonged pregnancy.
F E T A L A N D N E 0 N A T A L distress as a consequence of prolonged pregnancy long has been the subject of numerous studies. Previous investigations have focused mainly on the problems of placental insufficiency/· ~. 4 • H. 1n the postmaturity syndrome/· 4 • "'· 1 '· 1 ~· 31 and hypoglycemia in the neonate. 1 1. 22 On the other hand, little is known concerning the effects of prolonged pregnancy on the internal organs of the fetus. The present report is part of a series of electron microscopic investigations, with the rat used as an experimental model, to study the effects of prolonged pregnancy on var-
Material and methods
Twenty adult female Sprague-Dawley rats weighing 180 to 220 grams were used for this study. While in the proestrous phase, they were mated with male rats for 2 hours. The first day of pregnancy was designated as commencing 24 hours after spermatozoa were found in vaginal smears. Accordingly, the female rats deliver normally on Day 22 of pregnancy. The tissues examined consisted of livers from fetuses taken from 12 female rats whose gestational periods were prolonged 1, 2, or 3 days beyond term (Day 22). Prolonged pregnancy was achieved by daily subcutaneous injections of 7 mg. of progesterone*
From the Department of Anatomy, Faculty of Medicine, University of Manitoba. Supported by the Medical Research Council of Canada, Grant No. MA4740. Received for publication july 6, 1973. Revised September 6, 1973. Accepted September 17, 1973. Reprint requests: Dr. james A. Thliveris, Department of Anatomy, The University of Manitoba, 750 Bannatyne Ave., Winnipeg, Manitoba R3E OW3, Canada.
*Proluton, Schering Corp., lOll New Jerspy 07083.
864
Morri~
Aw., Vnion,
Ultrastructure of fetal liver
Volume 118 Number 6
in sesame oil from the twentieth through the twenty-fourth days of gestation. To evaluate any conceivable effect exogenous progesterone might have on fetal liver morphology, 4 pregnant female rats were given 7 mg. of progesterone daily from Day 14 through Day 21 of gestation. Fetal livers from this group were compared with livers of fetuses taken from normal term pregnancies. Laparotomy was performed on pregnant female rats under pentobarbital* anesthesia (5 mg. per 100 Gm. of body weight) on Days 22, 23, 24, and 25 (8 animals on Day 22 and 4 on each of the remaining 3 days) . Each fetal liver was removed after laparotomy, sectioned, and fixed for 2 hours at 4° C. in 3 per cent glutaraldehyde in O.lM phosphate buffer (pH 7.4). All tissues were rinsed for 24 hours at 4° C. in O.lM phosphate buffer (pH 7.4) containing 0.2M sucrose. Tissues were then postfixed for 2 hours at 4° C. in 2 per cent osmium tetroxide in O.lM phosphate buffer (pH 7.4). Following rapid dehydration in ascending concentrations of ethanol, the tissues were embedded in Epon 812. 13 Thick (1 p,) sections were stained with toluidine blue and examined for routine orientation. Thin sections were stained with uranyl acetate and lead citrate, viewed, and photographed in an electron microscope. t Results
Term (Day 22). At term, fetal rat hepatocytes were characterized by two discrete areas, one filled largely by glycogen and a second occupied by cytoplasmic organelles (Fig. 1). Glycogen was present mainly in the particulate or beta form, 15 but the larger rosettes or alpha particulates15 were also found (Fig. 2). Mitochondria varied in size and shape and contained lamellar cristae. The Golgi membranes and associated vesicles were well developed. Lysosomal-like
*Nembutal, Abbott Laboratories, 14th and Sheridan Rd., North Chicago, Illinois 60064. tEM 300, Philips Electronic Instruments, 750 S. Fulton Ave., Mount Vernon, New York 10550.
865
structures and microbodies were infrequently observed (Fig. 2) . The endoplasmic reticulum was predominately of the rough-surfaced type and was usually seen as parallel stacks of tubules, which frequently showed dilatations. Very few profiles of smooth reticulum were seen. Lipid droplets were also present. Prolonged gestation (Days 23, 24, and 25). The ultrastructure of fetal hepatocytes on Day 23 of gestation was similar to that described at term. On Days 24 and 25, several striking changes were present. Marked depletion of glycogen (Fig. 3) as well as the appearance of large quantities of smooth-surfaced endoplasmic reticulum was observed (Fig. 4). The smooth endoplasmic reticulum was always seen in association with the glycogen areas (Fig. 4). Communications between rough- and smoothsurfaced reticulum were frequently present (Fig. 5). The rough-surfaced endoplasmic reticulum did not appear altered from that seen at term. In addition, microbodies appeared more numerous on Days 24 and 25 than at term. Other organelles such as nuclei, Golgi complexes, and lysosomal-like structures did not appear altered. Greater numbers of hepatocytes exhibiting the above changes were seen on Day 25 rather than on Day 24. In contrast, while cells similar to those seen at term were detected on both days, fewer were observed on Day 25 than on Day 24. Fetal hepatocytes of preterm progesterone-treated female rats. The morphology of hepatocytes from fetuses of preterm progesterone-treated female rats was similar to that described for the term fetal hepatocytes. This observation ruled out an effect by progesterone on fetal hepatocyte morphology. In addition to the ultrastructural findings, fetal meconium staining, which is indicative of fetal distress, 4 was observed on Days 24 and 25 but not at term or on Day 23. Comment
The results of the present investigation have shown that glycogen was markedly de-
866
Thliveris Am .
1 1a rch 15, 197' Gy necol
J. O hst c·t.
Fig. 1. Fetal hepatocytes at term (Day 22 ) . These cells ·are characterized by glycogen-rich a reas ( Gl) and glycogen-free areas containing numerous cytoplasmic organelles. L , Lipid . ( Origina l magnification x7,400. )
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Ultrastructure of fetal liver
Fig. 2. Portion of a fetal hepatocyte at term (Day 22). Glycogen (GI) is present mainly in the particulate or beta form ( ~) . Larger rosettes or alpha particles are also seen (-I-+ ). Rough endoplasmic reticulum (RER) is abundant while smooth endoplasmic reticulum ( SER) is scarce. Mitochondria (M), Golgi apparatuses (G ), and microbodies (Mb) are shown. (Original magnification x19, 700.)
867
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Thliveris
March 15, 1974 Am. J, Obstet. Gynecol.
Fig. 3. Fetal hepatocytes on Day 24 of gestation. Hepatocytes at this stage show marked depletion of glycogen (Gl) and abundant smooth endoplasmic reticulum (SER). Increased numbers of microbodies ( Mb) are also seen. (Original magnification x7 ,400.)
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Ultrastructure of fetal liver
Fig. 4. Portion of a fetal hepatocyte on Day 24 of gestation. Numerous profiles of smooth endoplasmic reticulum (SER) are seen associated with glycogen (Gl) areas. Mitochondria (M) and rough endoplasmic reticulum (RER) are shown. (Original magnification x32,600.)
869
B70
Thliveris
Marclt 15, l9i4 Am. j. Obstet. Gynecol.
Fig. 5. Portion of a fetal hepatocyte showing communication between elements of the rough and smooth endoplasmic reticulum (arrows). Microbodies ( M) are shown. (Original magnification x26, 700.)
pleted from fetal hepatocytes during prolonged gestation (Days 24 and 25 ) . Concomitant with glycogen depletion was the appearance of large quantities of smooth endoplasmic reticulum which has been shown to be derived from rough endoplasmic reticulum.1~ Similar ultrastructural findings have been reported previously in hepatocytes of newborn rats"· n as well as in fasting adult rats.-' Moreover, associated with smooth endoplasmic reticulum is the enzyme glucose6-phosphatase which quantitatively increases during glycogenolysis. 1 " One might conclude that an inverse relationship existed between glycogen and smooth endoplasmic reticulum and glycogen and glucose-6-phosphatase as well as a direct relationship between smooth endoplasmic reticulum and the enzyme. However, Dawkins 7 has shown that during prolonged gestation in rabbits glucose-6-phosphatase did not increase but remained at the relatively low values present prior to birth. It is also known that the hepatic glycogen content in this species decreases substantially during prolonged pregnancy. 1n A marked increase in the enzyme level did occur 24 hours after delivery, the values being similar to those in 24-hour-old
rabbits delivered at term. Dawkins" has also provided evidence in rats that, while large amounts of hepatic glycogen were mobilized during the first 12 hours of life, the depletion was not dependent upon the magnitude of the rise in glucose-6-phosphatase. Ry utilization of inhibitors of protein synthesis, the normal high increase in enzyme activity was obstructed but not entirely prevented. Moreover, glycogen in addition to glucose-6phosphate levels in this condition decreased as readily as in untreated newborn animals. It would appear from the present investigation that while gJucose-6-phosphatase activity most likely did not increase during prolonged pregnancy the quantity present apparently was sufficient to allow glycogen mobilization to occur. The function of microbodies in hepatocytes remains unsettled, and the significance of the increased numbers of these structures observed in the present study is unknown. In prolonged pregnancy in man, fetal weight loss"· ' · 10 and an increased tendency toward neonatal hypoglycemia n. 12 occur iu those infants showing the postmaturity syndrome. Both of these conditions have been attrib11ted to placental insufficiency 1t is
Volume 118 Number 6
conceivable that if maternal nutrients were no longer adequately transferred by the placenta glycogen mobilization in the fetal liver possibly could sustain the fetus. The hypoglycemia present following delivery of a newborn infant in a prolonged gestation
Ultrastructure of fetal liver
871
might be partly because of a depletion of the large glycogen reserve, which under normal conditions at birth is readily mobilized in an attempt to maintain blood glucose levels until feeding comrnencesY
REFERENCES
1. Bleischer, N. A., Brown, J. B., and Townsend, L.: AM. J. OasTET. GYNECOL. 103: 496, 1969. 2. Browne, J. C. M.: AM. ]. 0BSTET. GYNECOL. 85: 573, !963. 3. Cardell, R. R.: Am. J. Anat. 131: 21, 1971. 4. Clifford, S. H.: J. Pediatr. 44: 1, 1954. 5. Dallner, G., Siekevitz, P., and Palade, G. E.: J. Cell Bioi. 30: 73, 1966. 6. Dallner, G., Siekevitz, P., and Palade, G. E.: J. Cell Bioi. 30: 9 7, 1966. 7. Dawkins, M. R. J.: Nature (Land.) 191: 72, 1961. 8. Dawkins, M. R. J.: Ann. N. Y. Acad. Sci. 111: 203, 1963. 9. Fischer-Rasmussen, W., Aegidius, J.: Acta Obstet. Gynecol. Scand. 51: 25, 1972. 10. Gruenwald, P.: AM. J. OaSTET. GYNECOL. 98: 503, 1964. 11. Khattab, A. K., and Forfur, J. 0.: Bioi. Neonat. 18: 1, 1971.
12. Leskes, A., Siekevitz, P., and Palade, G. E.: J. Cell Bioi. 49: 264, 1971. 13. Luft, J. H.: J. Biophys. Biochern. Cytol. 9: 409, 1961. 14. McKiddie, J. M.: Obstet. Gynecol. Survey 5: 44, 1950. 15. Revel, J. P.: J. Histochern. Cytochern. 12: !04, 1964. 16. Roux, J. F., Romrney, S. L., and Dinnerstein, A.: AM. J. OaSTET. GYNECOL. 90: 546, 1964. 17. Shelley, H. J.: Br. Med. Bull. 17: 137, 1961. 18. Sjostedt, S., Englesson, G., and Roath, G.: Arch. Dis. Child. 33: 123, 1958. 19. Strand, A.: Acta Obstet. Gynecol. Scand. (Suppl.) 45: 127, 1966. 20. Thliveris, J. A., and Connell, R. S.; Anat. Rec. 175: 607, 1973. · 21. Walker, J.: J. Obstet. Gynaecol. Brit. Ernp. 61: 162, 1954. 22. Zuspan, F. P.: J. Reprod. Med. 4: 77, 1970.