Difference between revisions of "Paper - Histological and histochemical observations on the corpus luteum"

From Embryology
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m (Observations on Corpra Lutea in Normal Pregnancy)
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Microscopic examination demonstrates that this corpus luteum is in no respect materially different from S48-2636 (figs. 2-4). K cells streaming into the granulosa lutein layer from the theca are prominent and numerous. Sudanophilic substances are much more prominent in the theca interna than in the granulosa lutein layer. Alkaline phosphatase is restricted to the theca interna and the endothelium of the blood vessels.
 
Microscopic examination demonstrates that this corpus luteum is in no respect materially different from S48-2636 (figs. 2-4). K cells streaming into the granulosa lutein layer from the theca are prominent and numerous. Sudanophilic substances are much more prominent in the theca interna than in the granulosa lutein layer. Alkaline phosphatase is restricted to the theca interna and the endothelium of the blood vessels.
  
A 413-Day Blm't0c_v5t, 19-Day Corpus Lzrteunz, 548-5000
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===A 4.5-Day Blastocyst, 19-Day Corpus Luteum, 548-5000===
  
 
This specimen is the first normal human blastocyst yet recovered from the uterine cavity. The associated endometrium is typical 19-day secretory endometrium.
 
This specimen is the first normal human blastocyst yet recovered from the uterine cavity. The associated endometrium is typical 19-day secretory endometrium.
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Microscopic examination reveals that this corpus luteum is almost identical in all respects with the 20-day corpus luteum of the normal menstrual cycle and with S48-3948, the corpus luteum associated with the abnormal 5-cell, 4‘/§_- to 5-day ovum.
 
Microscopic examination reveals that this corpus luteum is almost identical in all respects with the 20-day corpus luteum of the normal menstrual cycle and with S48-3948, the corpus luteum associated with the abnormal 5-cell, 4‘/§_- to 5-day ovum.
  
7‘/3- and 913-Day Pregrzazzcies
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===7.5- and 9.5-Day Pregnancies===
  
 
Figures 13 and 14, plate 3, are photomicrographs of sections of corpora ltttea associated with 7‘/3- and 9‘/3-day normal pregnancies respectively (Carnegie nos. 8020, 8215). Unfortunately, these sections, which were stained with haematoxylin and eosin, have faded
 
Figures 13 and 14, plate 3, are photomicrographs of sections of corpora ltttea associated with 7‘/3- and 9‘/3-day normal pregnancies respectively (Carnegie nos. 8020, 8215). Unfortunately, these sections, which were stained with haematoxylin and eosin, have faded
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to a great extent, but the following features are noteworthy. There is a progressive increase in vascularity and dilatation of the capillaries in the granulosa lutein layer. The theca lutein layer remains quite distinct and prominent. Peripheral vacuolization of the granulosa lutein cells, which is normally disappearing in the corpus luteum of the nonpregnant cycle by 26 to 27 days, becomes increasingly noticeable after 91/; days of pregnancy and is particularly striking at II and 12 days of pregnancy. The cell boundaries of the granulosa cells become progressively less distinct because of the line peripheral vacuolization. At this stage the granulosa cells resemble the “prickle cells” described by Hertig and Rock (1941). Although the K cells are not strikingly conspicuous in any of these sections, they are seen in considerable numbers. It appears that these cells have actually been stimulated and are assuming the appearance of full-blown activity noted in the 21-day corpus luteum (Hg. 7).
 
to a great extent, but the following features are noteworthy. There is a progressive increase in vascularity and dilatation of the capillaries in the granulosa lutein layer. The theca lutein layer remains quite distinct and prominent. Peripheral vacuolization of the granulosa lutein cells, which is normally disappearing in the corpus luteum of the nonpregnant cycle by 26 to 27 days, becomes increasingly noticeable after 91/; days of pregnancy and is particularly striking at II and 12 days of pregnancy. The cell boundaries of the granulosa cells become progressively less distinct because of the line peripheral vacuolization. At this stage the granulosa cells resemble the “prickle cells” described by Hertig and Rock (1941). Although the K cells are not strikingly conspicuous in any of these sections, they are seen in considerable numbers. It appears that these cells have actually been stimulated and are assuming the appearance of full-blown activity noted in the 21-day corpus luteum (Hg. 7).
  
A 12- to I 3-Day Pregnancy, Carnegie N 0. 8558, S46-2767
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===A 12- to 13-Day Pregnancy, Carnegie No. {{CE8558}}, S46-2767===
  
 
The corpus luteum was moderately cystic and measured 2.0 cm. in its greatest diameters. The convoluted border was 1 to 3 mm. thick and was pale yellow with an orange tinge, but did not appear to be senescent.
 
The corpus luteum was moderately cystic and measured 2.0 cm. in its greatest diameters. The convoluted border was 1 to 3 mm. thick and was pale yellow with an orange tinge, but did not appear to be senescent.
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A few of the theca lutein cells contain alkaline phosphatase, but most are devoid of the enzyme. A few K cells at the junction of the granulosa and theca lutein layers contain a high concentration of alkaline phosphatase, but of particular interest is the demonstration of the enzyme in significant amounts in some of the true granulosa lutein cells (fig. 17, pl. 3).
 
A few of the theca lutein cells contain alkaline phosphatase, but most are devoid of the enzyme. A few K cells at the junction of the granulosa and theca lutein layers contain a high concentration of alkaline phosphatase, but of particular interest is the demonstration of the enzyme in significant amounts in some of the true granulosa lutein cells (fig. 17, pl. 3).
  
A 16-Day Pregnancy, Carnegie No. 8602, S48-2088
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===A 16-Day Pregnancy, Carnegie No. {{CE8602}}, S48-2088===
  
 
The cystic corpus luteum measured 3 cm. in its greatest diameters. The convoluted borders were a brilliant yellow and varied in thickness from 4 mm. at the base to I mm. at the healed stigma.
 
The cystic corpus luteum measured 3 cm. in its greatest diameters. The convoluted borders were a brilliant yellow and varied in thickness from 4 mm. at the base to I mm. at the healed stigma.
 +
  
 
Haematoxylin and eosin sections of this specimen are very similar to those of the preceding stage. The theca interna is quite prominent (fig. 18, pl. 4). The granulosa lutein cells show considerable activity, manifested by noticeable peripheral vacuolization which causes cell boundaries to appear very indistinct. A large number of fine capillaries form a delicate vascular network in the granulosa lutein layer. K cells are even more numerous and distinct in this specimen than in the 12- to 13-day pregnancy (fig. 15), but a number of these cells are undergoing colloid degeneration as described in the corpus luteum of early menstruation. In this and in subsequent specimens to be described, a number of regular, spherical vacuoles are seen in the granulosa lutein layer. These vacuoles apparently mark the site of colloid deposits, which have dropped out of the section in preparation.
 
Haematoxylin and eosin sections of this specimen are very similar to those of the preceding stage. The theca interna is quite prominent (fig. 18, pl. 4). The granulosa lutein cells show considerable activity, manifested by noticeable peripheral vacuolization which causes cell boundaries to appear very indistinct. A large number of fine capillaries form a delicate vascular network in the granulosa lutein layer. K cells are even more numerous and distinct in this specimen than in the 12- to 13-day pregnancy (fig. 15), but a number of these cells are undergoing colloid degeneration as described in the corpus luteum of early menstruation. In this and in subsequent specimens to be described, a number of regular, spherical vacuoles are seen in the granulosa lutein layer. These vacuoles apparently mark the site of colloid deposits, which have dropped out of the section in preparation.
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In figure 20, plate 4, the theca lutein cells are seen to be almost completely devoid of alkaline phosphatase. Nearly all of the granulosa lutein cells, however, contain alkaline phosphatase in varying amounts, thereby making it difficult to identify the K cells. Several cells at the junction of theca lutein and granulosa
+
In figure 20, plate 4, the theca lutein cells are seen to be almost completely devoid of alkaline phosphatase. Nearly all of the granulosa lutein cells, however, contain alkaline phosphatase in varying amounts, thereby making it difficult to identify the K cells. Several cells at the junction of theca lutein and granulosa lutein layers contain a high concentration of the enzyme, and on the basis of their position and configuration these are very likely the K cells so conspicuously revealed by other techniques.
  
lutein layers contain a high concentration of the enzyme, and on the basis of their position and configuration these are very likely the K cells so conspicuously revealed by other techniques.
+
===A 26-Day Pregnancy, S48-263===
  
A 26-Day Pregnancy, S48-263:
+
This specimen was obtained with the uterus at time of operation for carcinoma of the cervix. The corpus luteum was cystic, 2 to 4 cm. in diameter, and was filled with a clear, yellowish fluid. The convoluted border was yellowish gray, and 2 to 3 mm. thick.
  
This specimen was obtained with the uterus at time of operation for carcinoma of the cervix. The corpus luteum was cystic, 2 to 4 cm. in diameter, and was filled with a clear, yellowish fluid. The convoluted border was yellowish gray, and 2 to 3 mm. thick.
 
  
 
The theca interna is very conspicuous in this specimen, being several cell layers thick around the entire granulosa lutein layer (fig. 21, pl. 4). The nuclei stain uniformly and present the appearance of actively functioning cells. For the first time since the stages of the mature follicle and the 16-day corpus luteum of the nonpregnant menstrual cycle, a number of small, irregular stellate cells with wrinkled hyperchromatic nuclei and homogeneous eosinophilic cytoplasm are seen in the theca lutein layer. Except for somewhat smaller size, these cells are identical in all respects with the K cells noted in routine haematoxylin and eosin sections of the younger corpora lutea. The sudden reappearance of these cells in the theca lutein layer is unexplained. There is no evidence of transformation or degeneration of theca lutein cells to these forms. The capillaries and sinusoids of the theca lutein layer are widely dilated.
 
The theca interna is very conspicuous in this specimen, being several cell layers thick around the entire granulosa lutein layer (fig. 21, pl. 4). The nuclei stain uniformly and present the appearance of actively functioning cells. For the first time since the stages of the mature follicle and the 16-day corpus luteum of the nonpregnant menstrual cycle, a number of small, irregular stellate cells with wrinkled hyperchromatic nuclei and homogeneous eosinophilic cytoplasm are seen in the theca lutein layer. Except for somewhat smaller size, these cells are identical in all respects with the K cells noted in routine haematoxylin and eosin sections of the younger corpora lutea. The sudden reappearance of these cells in the theca lutein layer is unexplained. There is no evidence of transformation or degeneration of theca lutein cells to these forms. The capillaries and sinusoids of the theca lutein layer are widely dilated.
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The granulosa lutein layer at this stage appears highly disorganized. Cell boundaries are quite indistinct. The individual granulosa lutein cells show marked variation in the stainability of their nuclei and cytoplasm. There are a large number of patent capillaries in the granulosa lutein layer. Around each of these capillaries, proliferation of perivascular connective-tissue elements is seen. There is a definite increase in the reticular network that at this stage encircles nearly every granulosa lutein cell. The central coagulum is well organized, and large, dilated vascular channels are present in this new connective tissue.
 
The granulosa lutein layer at this stage appears highly disorganized. Cell boundaries are quite indistinct. The individual granulosa lutein cells show marked variation in the stainability of their nuclei and cytoplasm. There are a large number of patent capillaries in the granulosa lutein layer. Around each of these capillaries, proliferation of perivascular connective-tissue elements is seen. There is a definite increase in the reticular network that at this stage encircles nearly every granulosa lutein cell. The central coagulum is well organized, and large, dilated vascular channels are present in this new connective tissue.
 +
  
 
Very few K cells that may be regarded as active are seen, but large numbers of these cells in all stages of degeneration are present. Colloid is present in larger amounts, and as a corollary the number of large, empty vacuoles is also increased over the preceding stage.  
 
Very few K cells that may be regarded as active are seen, but large numbers of these cells in all stages of degeneration are present. Colloid is present in larger amounts, and as a corollary the number of large, empty vacuoles is also increased over the preceding stage.  
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The theca interna is uniformly devoid of alkaline phosphatase (Hg. 23, pl. 4), although most of the granulosa lutein cells contain moderate amounts of the enzyme. It is diflicult to recognize K cells in this preparation. The endothelium of blood vessels in both layers contains alkaline phosphatase.
 
The theca interna is uniformly devoid of alkaline phosphatase (Hg. 23, pl. 4), although most of the granulosa lutein cells contain moderate amounts of the enzyme. It is diflicult to recognize K cells in this preparation. The endothelium of blood vessels in both layers contains alkaline phosphatase.
  
A 28- to 35-Day Preg.rzm1c_v, S48-4854
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===A 28- to 35-Day Pregnancy, S48-4854===
 +
 
 +
On gross examination the corpus luteum measured 2.3 by 2.0 cm. in its greatest diameters. The convoluted border was grayish yellow, measuring 2 to 3 mm. in thickness. The central coagulum was well organized.
  
On gross examination the corpus luteum measured 2.3 by 2.0 cm. in its greatest diameters. The convoluted border was grayish yellow, measuring 2 to 3 mm. in thickness. The central coagulum was well organizecl.
 
  
 
The theca lutein layer is still very prominent, although not so conspicuous as in the preceding specimen. Many of the nuclei are dense and irregularly pycnotic. The cytoplasm of most of the cells is highly vacuolated (fig. 24, pl. 5). The vascular sinusoids of the theca interna are almost completely collapsed.
 
The theca lutein layer is still very prominent, although not so conspicuous as in the preceding specimen. Many of the nuclei are dense and irregularly pycnotic. The cytoplasm of most of the cells is highly vacuolated (fig. 24, pl. 5). The vascular sinusoids of the theca interna are almost completely collapsed.
 +
  
 
The granulosa lutein layer presents essentially the same picture as that of the preceding specimen in haematoxylin and eosin preparations. Figure 25, plate 5, is a photomicrograph of this same section, showing a clearly defined colloid globule. Other K cells in various stages of degeneration are present in this section.
 
The granulosa lutein layer presents essentially the same picture as that of the preceding specimen in haematoxylin and eosin preparations. Figure 25, plate 5, is a photomicrograph of this same section, showing a clearly defined colloid globule. Other K cells in various stages of degeneration are present in this section.
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The alkaline phosphatase preparations are particularly striking. A number of the cells of the theca lutein layer contain large amounts of the enzyme, but most of the theca cells are totally devoid of it. On the other hand, the granulosa lutein layer contains a very high concentration of the enzyme, which is so diffusely distributed that it is impossible to separate the granulosa lutein cells from the K cells. Numerous large vacuoles, representing the site of colloid deposits that have been lost or dissolved in the process of preparation, are evident in this section as in figures 20 and 23.
 
The alkaline phosphatase preparations are particularly striking. A number of the cells of the theca lutein layer contain large amounts of the enzyme, but most of the theca cells are totally devoid of it. On the other hand, the granulosa lutein layer contains a very high concentration of the enzyme, which is so diffusely distributed that it is impossible to separate the granulosa lutein cells from the K cells. Numerous large vacuoles, representing the site of colloid deposits that have been lost or dissolved in the process of preparation, are evident in this section as in figures 20 and 23.
  
/I 4- to 4‘/_-;_.-Mont/z Pregrzrmcy, 548-2624
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=== A 4- to 4.5-Month Pregnancy, S48-2624===
  
 
This specimen was obtained incidentally to a total hysterectomy performed during the 5th month of pregnancy because of carcinoma of the cervix. The corpus luteum measured 2 by 1.5 cm. The stigma was depressed and well healed. The periphery of the corpus luteum presented a yellowish, fatlike appearance upon the cut surface. Several small gelatinous areas were seen between the convolutions. There was a small hemorrhage, 2 by 5 cm., in the exact center of the gland.
 
This specimen was obtained incidentally to a total hysterectomy performed during the 5th month of pregnancy because of carcinoma of the cervix. The corpus luteum measured 2 by 1.5 cm. The stigma was depressed and well healed. The periphery of the corpus luteum presented a yellowish, fatlike appearance upon the cut surface. Several small gelatinous areas were seen between the convolutions. There was a small hemorrhage, 2 by 5 cm., in the exact center of the gland.
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As in the haematoxylin and eosin preparations, the theca lutein layer is not evident in Sudan black treated sections. The granulosa lutein cells are uniformly devoid of lipid (fig. 28, pl. 5). An occasional K cell stands out conspicuously by virtue of its intense and uniform sudanophilia. Most of the K cells, however, appear only as “shadow forms,” having lost most
+
As in the haematoxylin and eosin preparations, the theca lutein layer is not evident in Sudan black treated sections. The granulosa lutein cells are uniformly devoid of lipid (fig. 28, pl. 5). An occasional K cell stands out conspicuously by virtue of its intense and uniform sudanophilia. Most of the K cells, however, appear only as “shadow forms,” having lost most of their sudanophilia. Though it is not evident in the photomicrograph, the fibrillar substructure of these cells is very conspicuous at higher magnification. Alkaline phosphatase is no longer demonstrable in the majority of granulosa lutein cells. A few cells along the outer margin of the lutein layer contain high concentrations of the enzyme (fig. 29, pl. 5). These cells occupy the same locus as the sudanophilic K cells mentioned above. It is of particular interest that the endothelium of blood vessels of both layers no longer contains demonstrable alkaline phosphatase.
 
 
of their sudanophilia. Though it is not evident in the photomicrograph, the fibrillar substructure of these cells is very conspicuous at higher magnification. Alkaline phosphatase is no longer demonstrable in the majority of granulosa lutein cells. A few cells along the outer margin of the lutein layer contain high concentrations of the enzyme (fig. 29, pl. 5). These cells occupy the same locus as the sudanophilic K cells mentioned above. It is of particular interest that the endothelium of blood vessels of both layers no longer contains demonstrable alkaline phosphatase.
 
  
 
==Observations on Corpora Lutea associated with Abnormal Ova==
 
==Observations on Corpora Lutea associated with Abnormal Ova==

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White RF. Hertig AT. Rock J. and Adams E. Histological and histochemical observations on the corpus luteum of human pregnancy with special reference to corpora lutea associated with early normal and abnormal ova. (1951) Contributions To Embryology, No. 224 57-71.

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This 1951 paper is a historic histological study of the development of the corpus luteum.


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Histological and Histochemical Observations on the Corpus Luteum of Human Pregnancy with special reference to Corpora Lutea associated with early Normal and Abnormal Ova

White RF. Hertig AT. Rock J. and Adams E.

Free’ I-Io.s'pz'ral for I-Vomcw, Brool(1inc,' Dcpmwnxcvzts of Pathology, O[25tctric5, and Gynecology, Hm'z2ard Medical School

With seven plates

Review of the Literature

Speculation and investigation concerning the role of the human corpus luteum in the normal menstrual cycle and in pregnancy have occupied the energies of many investigators since early times. For excellent reviews of the work that has led to modern concepts of the corpus luteum, the reader is referred to Asdell (1928) and Pratt (1935).


Meyer (1911) was the first to describe in detail the macroscopic and microscopic appearance of the stages from the ruptured follicle to complete involution of the human corpus luteum. He was the first to point out that there are four recognizable stages in the development of the corpus luteum, namely proliferation, vascularization, mature or so-called blossom stage, and regression. For the sake of completeness, Meyer’s concept of the development of the corpus luteum, which has been generally accepted by workers in the field, will be outlined briefly.


During the proliferative stage, which follows immediately upon rupture of the mature Graafian follicle, there is increased vascularity of the theca interna and theca externa, evidenced by dilatation of the capillaries. Storage of fat occurs in both theca interna and granulosa, but more noticeably in the former; mitoses in the granulosa layer are less evident or entirely absent, but mitotic figures are still present in the theca interna; hemorrhage into the follicle is not the rule, because capillaries do not extend into the granulosa layer at or before rupture. The stage of vascularization is marked by a well defined membrana granulosa which has increased conspicuously by hypertrophy of the former granulosa cells. The theca externa is still quite evident, but the cells of the theca interna are already shrunken and are no longer coherent. The folding of the wall of the corpus luteum is much more marked, and there is a notable increase of storage of lipoid in the granulosa cells, which may be termed granulosa lutein cells at this stage of development. Fibroblasts are found in the central coagulum as early as 48 hours after rupture of the follicle.


As the corpus luteum matures and attains the socalled blossom stage, the cells of the theca interna are irregular and decreased in number. The uniform granulosa lutein cells are large and irregularly cuboidal, with increased lipoid content. Capillaries, accompanied by a few spindle cells, pass between the luteal cells, and individual luteal cells are surrounded by connective-tissue fibrils. The inner contour of the granulosa lutein layer is lined smoothly by a conspicuous capillary network. The central coagulum has undergone considerable connective-tissue organization, or it may become cystic, or a hematoma may form in it. Unless pregnancy is superimposed, the corpus luteum goes on to regression, which is characterized by fatty degeneration and simple atrophy of lutein cells, associated with increased invasion of the luteal tissue by connective-tissue elements. Ultimately, the lipoid substance of the lutein tissue disappears and the corpus luteum is transformed into the corpus albicans by a process of hyaline degeneration which may extend over several months.


Meyer (1911, 1932) observed that the mature stage and the regressive stage were imperfectly limited and beginning regression could not be definitely recognized. Novak (1932, 1941) stated that regression begins shortly before menstruation, about the 26th day of a normal 28-day cycle. Brewer (1942), however, presents evidence that regression begins at the termination of the vascular stage, about 4 to 6 days before the onset of menstruation. Chemical assays of lutein tissue reveal a steady increase in phospholipid from immediately after ovulation until the 10th day, after which time the phospholipid content of the gland falls. During the first to days of existence of the corpus luteum there is a slight fall in the cholesterol ester content of lutein tissue, but after this time there is an abrupt increase in cholesterol ester content (Brewer, 1942). These demonstrable chemical changes in the corpus luteum, together with the microscopic appearance of the gland, led Brewer to conclude that the corpus luteum commences to regress about 8 to 10 days after ovulation.


Gillman and Stein (1941) tabulate the number of corpora lutea of pregnancy examined by various investigators prior to 1941, including their own series of 19 specimens. Brewer (1942) reports examining 8 corpora lutea of early pregnancy, but does not furnish any details concerning the histology of these specimens. I-Iertig and Rock (1941, 1945, 1949a) and Heuser, Rock, and Hertig (1945) describe briefly 8 corpora lutea associated with early pregnancies ranging from 7 days ovulation age to the definitive yolksac stage.


Meyer (1911) characterized the corpus luteum of pregnancy from the 2d month of pregnancy on as having a coarser, cruder appearance due to hypertrophy and increased lipid deposition in the lutein cells, a high degree of connective-tissue proliferation around and between the luteal cells, and thick-walled capillaries. He noted that hyaline degeneration in the corpus luteum of pregnancy was long delayed. He remarked that the theca lutein cells are sometimes retained up to the 4th month of pregnancy, but are insignificant and decrease in number until the final month of pregnancy, when the theca cells flourish again.


Gillman and Stein (1941), in a study of 13 corpora lutea associated with intra-uterine pregnancies ranging froi 35 to 282 days, state that there is a “critical period” of sudden growth in the volume of the corpus luteum between the 50th and 60th days of pregnancy, due to an excess production of hormonal fluid in the fibrotts-tissue-lined cavity, which is subsequently obliterated; that the theca lutein cells attain their maximum development synchronously with the development of this cavity, and that after its collapse they also disappear; that the granulosa cells, on the other hand, persist throughout pregnancy, and that their vacuolar secretion may degenerate into colloid or even calcium-containing deposit.


Brewer (1942) noted, in addition to sustained high phospholipid levels and low cholesterol ester levels in the corpus luteum of pregnancy, an increase in vascularity, a lack of fatty degeneration, and absence of simple atroppy of the luteal cells.


Asdell (1928) and Pratt (I935) have reviewed the literature concerning the origin of the true lutein cell of the human corpus luteum. Meyer decided that the theca lutein cells disappear early in the life of the corpus luteum, leaving only luteal cells of granulosa origin. Chydenius (1926) decided upon a dual origin of the lutein cell. Shaw (1925) described theca lutein cells, or paralutein cells as he termed them, but considered that they remain at the periphery of the gland and do not take part in the formation of the stroma of the gland. A real dilierence between species does seem to exist in the extent to which theca lutein cells invade the stroma of the corpus luteum. According to Hammond and Marshall (1925), this invasion appears to be small or entirely wanting in the monotremes, marsupials, certain rodents, the sheep, the horse, and man, whereas in bats, the guinea pig, the cow, and most of all the sow, the invasion appears to be considerable.


McNutt (1924) asserted the dual origin of the lutein cell in the cow, stating that small clumps of theca lutein cells invade the space filled by the granulosa lutein cells and become detached from the connectivetissue framework. Despite some divergence of opinion regarding the origin of the human lutein cell, it is established that early in the development of the corpus luteum, the lutein cell derived from the membrana granulosa becomes dominant and is the cell usually described as the lutein cell (Pratt, 1935).


Corner (1915) described, in addition to the granulosa lutein and theca lutein cells, cells of a third type in the corpus luteum of the sow. These are smaller than the lutein cell, of varied shape, and strongly eosinophilic. They contain small vacuoles even when fixed with osmic acid. He believed that Delestre (1910) also saw them. Gillman and Stein (1941) pointed out the existence of dark and light cells in the granulosa and regarded them as representing different phases of activity of secretion in granulosa lutein cells.


In recent years a number of new methods of chemical cytology have been developed, and these are being applied to a great variety of organs and tissues. An excellent review of advances in this field is given by Dempsey (1948). To date, reports of the application of these techniques to the human corpus luteum are few.


McKay and Robinson (1947) studied a series of human corpora lutea of the normal menstrual cycle, employing some of the newer techniques for detecting presumptive ketosteroid compounds. The presence of birefringent crystals, autofluorescent materials, acetone-soluble keto compounds, and sterol substances which reacted with sulfuric acid was noted. ‘Nhen employing Sudan IV to detect sudanophilie lipids, they noticed that lipid was confined to the theca interna in the developing follicle. In the 15-day corpus luteum, small sudanophilie drops were found in all the granulosa and theca lutein cells. During the next 6 or 7 days of the life of the corpus luteum, fine peripherally distributed lipid droplets were seen in almost all the granulosa cells. During this period there was a steady increase in the number and size of sudanophilie droplets in the cells of the theca interna, these droplets being much larger than those in the granulosa cells. About the 23cl day of the cycle a marked decrease was observed in the number of sudanophilie granules in the granulosa lutein cells, many cells being completely devoid of lipid. There were, however, patchy areas containing large drops of lipid in the granulosa layer around the blood vessels of the invading connective-tissue septa. Fewer theca interna cells contained lipid than in earlier stages, but those that did, contained large droplets. This decrease in total sudanophilie substance in both layers was maintained to the 27th day of the cycle. After the 27th day, a greatly increased amount of sudanophilie substance distributed in large, coarse droplets was observed in both layers.


Examination for birefringent materials revealed a few tiny anisotropic crystals in the theca interna of the 15-day corpus luteum. There was a steady increase in the size and number of these crystals up to the 24th day of the cycle. Very fine birefringent crystals were seen in the granulosa cells on the 17th day, increasing in number and size until the 22d day of the cycle. Throughout the period embracing the 17th to the 23d day of the menstrual cycle, more anisotropic substance was present in the theca interna than in the granulosa at any stage of development. There was a notable decrease in the amount of hirefringent crystals in the theca interna from the 24th to the 27th day, after which there was a considerable increase that persisted through menstruation. A comparative absence of birefringent crystals was observed in the granulosa lutein layer from the 22d day through late menstruation.


Studies of autofluorescent substances and substances that reacted with phenylhydrazine and sulfuric acid essentially paralleled the observations made on birefringence.

McKay and Robinson (1947) also noted in the granulosa layer a few extracellular large oily drops that were sudanophilie and reacted with sulfuric acid and phenylhydrazine but were negative for birefringence and autofluorescence. These were seen only after the 22d day of the cycle, when reactive materials had disappeared from the granulosa cells. The authors suggest that these large drops may be indicative of a change from active secretion to storage or inactivity, basing this interpretation on findings in the adrenal gland (Selye, 1937; Sarason, 1943; Dalton et al., 1944) that line lipid droplets are associated with active secretion and large droplets with inactivity.


Corner (1948) in a study of 3 human corpora lutea observed that the cells of the theca interna contain alkaline phosphatase up to a day or two after ovulation, but subsequently lose it. The granulosa lutein cells seemed to be almost devoid of alkaline phosphatase in the stages studied.

It is the purpose of this paper to describe in some detail the histological and histochemical variations of the human corpus luteum from the earliest stage of pregnancy yet obtained, a 2-cell tubal ovum (Hertig and Rock, 1949b), to that associated with a 4.5 month fetus. For purposes of comparison, a study of corpora lutea of the normal menstrual cycle is included. Evidence will be presented that the lutein cell is derived from the membrana granulosa of the follicle. A third cell type, derived from the theca interna of the follicle, has been demonstrated. Cells of this type make their way into the granulosa layer at or shortly after the time of rupture of the follicle, and become quite prominent as the gland approaches its peak of physiological activity. Evidence will be presented that these cells are the site of intense localization of ltetosteroids, which presumably are the secretory products of the corpus luteum.

Materials and Methods

Since 1939 two of the authors (I-Iertig and Rock) have been searching for early human ova, and to date they have succeeded in obtaining 32 ova ranging from 2 to 17 days ovulation age. Of these specimens, 19 are regarded as normal. The remaining 13 were adjudged to be abnormal because of one or more of the following conditions: multinucleated blastomeres, shallow implantation, malorientation or lack of germ disk, defective trophoblast, or abnormal segmentation cavity. These specimens were obtained as described by Hertig and Rock (1944). In all instances, the ovary containing the corpus luteum was removed with the uterus at operation. The corpora lutea thus obtained were examined macroscopically and then appropriately prepared for microscopic examination as described below.


Nine corpora lutea associated with older pregnancies 25 days to 4.5 months) were also obtained from surgical material from the Free Hospital for Women (Brookline, Massachusetts) or the Boston Lying-In Hospital.


Forty-eight corpora lutea not associated with pregnancy were studied in order to compare histologic and histochemical changes in the gland during the normal menstrual cycle with those observed in pregnancy.


All corpora lutea of nonpregnant cycles and those associated with ova still free in the tubes or uterine cavity were dated by referring to the endometrial age (Hertig, 1945). It is assumed that the normal menstrual cycle is 28 (lays in length, with ovulation on the 14th day of the cycle. It is generally agreed that, regardless of the length of the menstrual cycle, ovulation takes place about 14 days (:2 (lays) before the first day of the next expected period (Rock and Hertig, 1944).


Material obtained prior to June 1947 had been fixed and stained in accordance with routine laboratory procedures, the following stains being employed: haematoxylin and eosin, eosin-methylene blue, ironalum haematoxylin, phosphotungstic acid-haematoxylin, and Scarlet red for fats. Recently acquired specimens have been stained with eosin-methylene


blue and with haematoxylin and eosin, and have been subjected to the following special procedures: The alkaline phosphatase procedure of Gomori (1941) was applied to all recently acquired corpora lutea. Frozen sections, 10 to 20 microns in thickness, of formalin-fixed material were prepared, and alternate sections were treated with acetone, alcohol, and acetone—alcohol mixtures for comparison with untreated sections. Sudan black was used as a general lat stain. The phenylhydrazine reaction of Bennett (1940) and the plasmal reaction as described by Lison (1936) were applied to representative cases. Plain sections, both untreated and subjected to fat solvents, were mounted in glycerine jelly and examined under crossed Nicol prisms for evidence of birefringence; and under a fluorescence microscope similar to that described by Grafllin (1939), using as illumination a beam of ultraviolet rays obtained by filtering the light of a carbon-arc lamp through a copper sulfate solution and a Corex filter no. 586.


Formalin-fixed frozen sections were floated onto slides and blotted dry, and a drop of a solution consisting of equal parts of concentrated sulfuric acid and acetic anhydride was put on the tissue. The slides were examined immediately for the appearance of brown droplets in the cells of the corpus luteum and ovarian stroma. This reaction, formerly used as a test for cholesterol (Romeis, 1928), is now ltnown to depend upon the presence of unsaturated bonds in steroid molecules (S0l)0tl{:1, 193,8).


Very recently new techniques for the histochemieal detection of active carbonyl groups in lipoid and nonlipoitl materials have been developed (Seligman and Ashbel, I949; Ashbel and Seligman, 1949), and through the courtesy of Drs. A. M. Seligman and R. Ashbel, of the Beth Israel Hospital, Boston, Massachusetts, selected corpora lutea were studied employing these new techniques.


Two specimens were treated for the histochemieal demonstration of phospholipid according to the method of Baker (1946).

Observations on Corpra Lutea of the Nonpregnant Menstrual Cycle

The Mature Grcmfirm Follicle, S48-731

Figure 1, plate I, is a photomicrograph of a typical mature Graafian follicle. The theca interim is several cell layers thick. Many of the theca cells are highly vacuolated. Widelyr dilated, blood—filled capillaries are prominent in the theca immediately beneath the basal layer of the granulosa. i\/Iitotic figures are rare in the theca interna. Conspicuous in the theca are a number of cells that differ markedly from the neighboring theca cells. These cells have small, dense, hyperchromatic nuclei which are irregular in outline, and stand out in bold relief against the nuclei of the theca interna cells, which by contrast are plump, ovoid, and vesicular, with a single prominent, eccentric nucleolus. The cytoplasm of these stellate cells is homogeneous and more strikingly eosinophilic than that of the theca cells. Careful study has shown that these cells do not represent intravascular or perivascular elements. For lack of a more specific and descriptive name, and in the interest of definiteness without repetition, these cells will be referred to in the subsequent descriptions and discussions as “K cells.”


The membrana granulosa is sharply demarcated from the theca interna by a closely packed layer of cells contiguous with, and similar to, the cells of the membrana itself, which is many cell layers thick and thrown into convolutions by the hypertrophy and multiplication of the granulosa cells. Consequently, the inner layer of the follicle presents a scalloped, undulating edge, with tongues of theca interna projecting into the convolutions from the stroma of the ovary. Mitotic figures are numerous in the membrana granulosa. The nuclei of the granulosa cells are perhaps a little larger than those of the theca interna cells, and because of their rapid rate of division and growth are more basophilic. Their cytoplasm presents a frothy appearance, and cell outlines are indistinct, although there is no evidence of vacuolization. There is no evidence of capillary penetration into or proliferation within the membrana granulosa. None of the K cells present in the theca interna are seen in the membrana granulosa at this stage of development.


Corpus Luteum of 16th Day of Cycle, Estimated Age 2 Days, S48-2636

Macroscopically, this corpus luteum appeared as a hemorrhagic, unhealed crater I cm. in diameter and 2 mm. in depth, on the posterior surface of the ovary.

Haematoxylin and eosin sections (fig. 2, pl. 1) show the theca interna to be considerably thinned out and the membrana granulosa thrown into a large number of deep convolutions, owing in part to the collapse of the follicle after rupture. The vascular channels of the theca interna are widely dilated, and in several places endothelial sprouts are seen penetrating the membrana granulosa from the theca interna. The cells of the theca interna are essentially the same as those observed in the mature follicle, and rarely show mitoses. The granulosa cells are plump and polyhedral, with round, vesicular nuclei. Their cytoplasm is frothy, but no distinct vacuolization is seen. The granulosa cells are arranged in bundles or fascicles separated in many areas by large lacunae of extravasated blood. These lacunae are not lined with endothelium.

The K cells noted in the theca interna of the mature follicle are very prominent at this stage of development of the corpus luteum. Although few of them are found in the theca interna, ribbons of them can be seen spreading out into the membrana granulosa, penetrating as far as the central coagulum. The attenuated cytoplasmic processes of these cells suggest amoeboid activity. It is to be noted that these cells are more numerous at this and subsequent stages of development than in the mature follicle. Only once have we observed a mitotic figure in a cell that we could definitely say was a K cell. It is possible that the small, irregular, hyperchromatic nuclei characteristic of these cells at this stage represent rapid mitotic activity. As will be noted later, the nuclei of these cells become larger and less hyperchromatic as the gland approaches the period of maximum functional activity.


Sudan black preparations (fig. 3, pl. 1) reveal that the cells of the theca interna contain much more lipid than do those of the membrana granulosa. This lipid is distributed in fine droplets in most cells, although medium-sized and coarse droplets are found in some of the theca interna cells. As has been mentioned, the granulosa cells contain much less lipid, and this is evenly distributed as a fine peripheral dusting of sudanophilic lipid. In an occasional granulosa cell a few coarse globules of lipid are seen. The K cells are distinguishable only with some diliiculty. They present a uniform, nongranular sudanophilia.


Alkaline phosphatase is localized exclusively in the cells of the theca interim and in the endothelium of blood vessels. The cytoplasm of the granulosa cells contains no alkaline phosphatase at this stage of development (fig. 4, pl. 1).

Corpus Luteum of 20th Day of Cycle, Estimated Age 6 Days, S48-2262

On cut section, the corpus luteum measured 1.7 by 1.5 cm. in its greater diameters. The convoluted borders were bright yellow and from 1 to 3 mm. thick. The central coagulum was pale gray with several small hemorrhagic areas.

The corpus luteum at this stage is quite compact. There is very little extravasated blood in either layer. The blood vessels of the theca interna are widely dilated. Definite capillaries are seen in the granulosa layer, but these are still small and only an occasional The central coagulum contains many proliferating fibroblasts and red blood cell can be noted in them.

thus is undergoing early organization. No capillaries are seen in it. The cells of the theca interna appear to be widely separated by fibroblasts and other connective—tissue elements; their cytoplasm is markedly vacuolated, but the nuclei show no evidence of cellular degeneration. The granulosa cells are large and polyhedral, with distinct cell membranes and marked peripheral vacuolization of the cytoplasm (fig. 5, pl. 1). Interspersed among the granulosa cells are a number of K cells, whose nuclei appear dark, irregular, and almost pycnotic.


Sudanophilic substance is distributed irregularly in the cells of the theca interna. Some of the theca cells contain few lipid droplets; others contain numerous fine, peripherally distributed lipid droplets, and many contain large, coarse globules of sudanophilic substances. Nearly all the granulosa lutein cells contain fine, peripherally distributed lipid droplets. Only an occasional granulosa cell contains the coarse lipid droplets noted in the theca. The large, stellate K cells are particularly conspicuous at this stage. All of them appear uniformly sudanophilic, although close examination reveals some granular deposits of sttdanophilic substances which are almost masked by the uniformly sudanophilic background of the cytoplasm of these cells (fin. 6, pl. 1). The nuclei of the K cells are clear and devoid of lipid.


The theca lutein cells are uniformly devoid of alkaline phosphatase. This statement applies equally well to the majority of the granulosa cells. A small number of cells scattered among the granulosa cells, however, contain alkaline phosphatase in moderate amounts. It is difiicult to state at this stage of development that these cells containing alkaline phosphatase deposits are the K cells so prominent in the Sudan black preparation.


Corpus Luteum of 23rd Day of Cycle, Estinzazerl Age 9 Days, S48-3028

The corpus luteum measured 1.5 cm. in its greatest diameters. The convoluted border was bright yellow, and averaged 3 mm. in thickness. The central coagt1him was well organized and was the site of a recent small hemorrhage.

It is to be noted in this specimen that the theca lutein layer is much less prominent than in the 20-day corpus luteum, but that those theca lutein cells that are observed present essentially the same characteristics as those noted in the earlier stage. The granulosa lutein cells are plump and show all the evidences of marked physiological activity. The K cells are quite numerous and prominent in this specimen and are readily recognized by their more angular shapes, dark, homogeneous cytoplasm, and small, dark nuclei (Fig. 7, pl. 2). Capillaries in the granulosa lutein layer are numerous and widely dilated.


The theca lutein cells contain large, coarse drops of lipid, whereas the granulosa lutein cells contain fine, peripherally distributed lipid granules in large numbers (fig. 8, pl. 2). The K cells are especially prominent in this preparation, and there can be little question that these elongated, uniformly sudanophilic cells with clear, ovoid nuclei are the same cells that are so conspicuous in haemato:~:ylin and eosin preparations.


Only an occasional theca lutein cell contains alkaline phosphatase (fig. 9, pl. 2). The true granulosa lutein cells are devoid of this enzyme. A number of cells, however, having the configuration and nuclear characteristics of the K cells as they appear in routine and Sudan-treated preparations contain high concentrations of alkaline phosphatase. Capillary endothelium in all layers of the corpus luteum contains the enzyme in high concentration.

From 24th Day of Cycle to Menstuation

Between the 23d day of the cycle and the onset of menstruation, the evidence of regression in the corpus luteum becomes increasingly marked. There is no evidence of further capillary proliferation. The theca cells become less and less distinct, until they can be found only in widely separated clumps in the connective-tissue septa invaginating the granulosa from the ovarian stroma. The granulosa lutein cells show evidence of degeneration, manifested by loss of chromaticity of the nuclei and loss of most of the peripheral vacuolization noted at earlier stages. Many granulosa cells show increasing accumulation of medium—sized and coarse lipid droplets.

The fate of the K cells becomes evident during this period. With haematoxylin and eosin stains these cells become more eosinophilie, the cytoplasm becomes more dense and homogeneous, and the nuclei become quite contracted and hyperchromatic. The cytoplasm seems to condense, until ultimately all that remains of many of these cells late in the life of the corpus luteum is a dense, eosinophilie mass similar to the colloid described by Gillman and Stein (1941). This process of colloid degeneration evidently extends over a long period, because, as will be noted in our discussion of the corpus luteum of early menstruation, large numbers of apparently normal K cells can be found in older corpora lutea. The K cells are still prominent in Sudan black preparations, but show a progressive loss of sudanophilia and a retraction of their cytoplasmic processes. The pattern of alkaline phosphatase distribution remains essentially the same as that observed in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells as the corpus luteum becomes older.

Corpus Luteum during Early Menstruation, S48-535

At this stage the theca interna is very indistinct. True theca lutein cells are found only in scattered clumps, chiefly in the connective-tissue septa that penetrate between the folds of the granulosa from the ovarian side of the gland (fig. 10, pl. 2). Those theca cells that persist are markedly vacuolated and show wide variation in the staining characteristics of their nuclei. Most of the granulosa lutein cells have lost the peripheral vacuolization that typifies the actively secreting gland. Many cell boundaries in the granulosa lutein layer are quite indistinct. The capillaries in both cell layers are more or less uniformly collapsed, and contain few erythrocytes. Although the K cells are still prominent in routine sections, they do not present the full-blown appearance seen in the 23-day corpus luteum. Moreover, their cytoplasm is denser than has previously been noted and in most instances appears to be contracting, leaving large vacuoles between the granulosa lutein cells. Some of the K cells have degenerated to what we consider the end stage of this line of cells, a dense, strongly eosinophilie colloid droplet. All gradations between a stellate, active cell and the final degenerative end product, colloid, can be found in this specimen, a fact which indicates that this process of colloid degeneration must extend well beyond the menstrual period.


The theca lutein cells contain only medium-sized and coarsely irregular lipid droplets (fig. 11, pl. 2). The true granulosa cells contain much more lipid than at any previous stage. Although the lipid droplets are larger than in earlier stages, they are peripherally distributed in most granulosa cells, and in none are the droplets as large and coarse as those in the theca lutein cells. A few granulosa cells are heavily laden with medium-sized lipid droplets. The K cells are still prominent in the Sudan black preparations, but their cytoplasmic processes are markedly retracted, the cytoplasm has lost some of its uniform homogeneous sudanophilia, and a few fine sudanophilic granules are seen against the slate-gray cytoplasmic background of the cells. A few large, oily drops showing varying degrees of sudanophilia are present. It is believed that these represent the colloid drops noted in the haematoxylin and eosin preparations.

The alkaline phosphatase preparations show essentially the same pattern of distribution of the enzyme as in the 23-day specimen, with perhaps some diminution in the concentration of the enzyme in the K cells (fig. 12, pl. 2).

Observations on Corpra Lutea in Normal Pregnancy

A 2-Cell Egg, 17-Day Corpus Luteum, S49-2439

This ovum was recovered from the middle third of the Fallopian tube and consisted of two normal blastomeres. This specimen, of about 60 hours coital age, is the earliest human ovum yet recovered. The endometrium had the characteristics of that of 2 to 21/; days after ovulation.

On cut surface, the corpus luteum measured 2.5 by 1.5 cm. in its greatest diameters. The convoluted border was red-gray, and varied in thickness from 3 mm. at the base to I mm. at the unhealed stigma. The central coagulum was moderately well organized and showed some peripheral congestion.

Microscopic examination demonstrates that this corpus luteum is in no respect materially different from S48-2636 (figs. 2-4). K cells streaming into the granulosa lutein layer from the theca are prominent and numerous. Sudanophilic substances are much more prominent in the theca interna than in the granulosa lutein layer. Alkaline phosphatase is restricted to the theca interna and the endothelium of the blood vessels.

A 4.5-Day Blastocyst, 19-Day Corpus Luteum, 548-5000

This specimen is the first normal human blastocyst yet recovered from the uterine cavity. The associated endometrium is typical 19-day secretory endometrium.

On cut surface the corpus luteum measured 2.2 cm. in its greatest diameters. The convoluted borders were reddish gray, measuring 3 to 4 mm. in thickness. The stigma was completely healed, and the central coagulum was pale and gelatinous.

Microscopic examination reveals that this corpus luteum is almost identical in all respects with the 20-day corpus luteum of the normal menstrual cycle and with S48-3948, the corpus luteum associated with the abnormal 5-cell, 4‘/§_- to 5-day ovum.

7.5- and 9.5-Day Pregnancies

Figures 13 and 14, plate 3, are photomicrographs of sections of corpora ltttea associated with 7‘/3- and 9‘/3-day normal pregnancies respectively (Carnegie nos. 8020, 8215). Unfortunately, these sections, which were stained with haematoxylin and eosin, have faded

to a great extent, but the following features are noteworthy. There is a progressive increase in vascularity and dilatation of the capillaries in the granulosa lutein layer. The theca lutein layer remains quite distinct and prominent. Peripheral vacuolization of the granulosa lutein cells, which is normally disappearing in the corpus luteum of the nonpregnant cycle by 26 to 27 days, becomes increasingly noticeable after 91/; days of pregnancy and is particularly striking at II and 12 days of pregnancy. The cell boundaries of the granulosa cells become progressively less distinct because of the line peripheral vacuolization. At this stage the granulosa cells resemble the “prickle cells” described by Hertig and Rock (1941). Although the K cells are not strikingly conspicuous in any of these sections, they are seen in considerable numbers. It appears that these cells have actually been stimulated and are assuming the appearance of full-blown activity noted in the 21-day corpus luteum (Hg. 7).

A 12- to 13-Day Pregnancy, Carnegie No. 8558, S46-2767

The corpus luteum was moderately cystic and measured 2.0 cm. in its greatest diameters. The convoluted border was 1 to 3 mm. thick and was pale yellow with an orange tinge, but did not appear to be senescent.

The theca lutein layer is quite prominent and many cell layers in thickness. The cytoplasm of these cells is vacuolated, and the nuclei resemble those of actively functioning cells. The granulosa lutein cells show marked peripheral vacuolization, and the cell boundaries are thus almost completely obliterated (fig. 15, pl. 3). There has been a significant increase in the number of widely patent vascular channels in the granulosa layer. K cells are not at all conspicuous in haematoxylin and eosin sections. However, the dense, pycnotic nuclei and contracted cytoplasm characteristic of many of the K cells in figure 10 are not frequently seen here, a fact which indicates a recrudescence of activity of these cells.

A very striking change is noted in the Sudan black preparations. Only a few theca interna cells contain coarse lipid granules. The majority of the theca lutein and granulosa cells contain large numbers of very fine, peripherally distributed sudanophilic droplets.

K cells are very prominent (fig. 16, pl. 3) and show marked uniform sudanophilia and attenuated cytoplasmic processes like those seen in the physiologically active corpus luteum of the 23d clay of the nonpregnant cycle (fig. 8).

A few of the theca lutein cells contain alkaline phosphatase, but most are devoid of the enzyme. A few K cells at the junction of the granulosa and theca lutein layers contain a high concentration of alkaline phosphatase, but of particular interest is the demonstration of the enzyme in significant amounts in some of the true granulosa lutein cells (fig. 17, pl. 3).

A 16-Day Pregnancy, Carnegie No. 8602, S48-2088

The cystic corpus luteum measured 3 cm. in its greatest diameters. The convoluted borders were a brilliant yellow and varied in thickness from 4 mm. at the base to I mm. at the healed stigma.


Haematoxylin and eosin sections of this specimen are very similar to those of the preceding stage. The theca interna is quite prominent (fig. 18, pl. 4). The granulosa lutein cells show considerable activity, manifested by noticeable peripheral vacuolization which causes cell boundaries to appear very indistinct. A large number of fine capillaries form a delicate vascular network in the granulosa lutein layer. K cells are even more numerous and distinct in this specimen than in the 12- to 13-day pregnancy (fig. 15), but a number of these cells are undergoing colloid degeneration as described in the corpus luteum of early menstruation. In this and in subsequent specimens to be described, a number of regular, spherical vacuoles are seen in the granulosa lutein layer. These vacuoles apparently mark the site of colloid deposits, which have dropped out of the section in preparation.


The great majority of both theca lutein cells and granulosa lutein cells contain large numbers of fine peripheral sudanophilic droplets. An occasional theca lutein cell contains a number of medium-sized lipid droplets (fig. 19, pl. 4). The K cells are quite conspicuous and appear essentially the same as in the corpus luteum of the 12- to 13-day pregnancy.


In figure 20, plate 4, the theca lutein cells are seen to be almost completely devoid of alkaline phosphatase. Nearly all of the granulosa lutein cells, however, contain alkaline phosphatase in varying amounts, thereby making it difficult to identify the K cells. Several cells at the junction of theca lutein and granulosa lutein layers contain a high concentration of the enzyme, and on the basis of their position and configuration these are very likely the K cells so conspicuously revealed by other techniques.

A 26-Day Pregnancy, S48-263

This specimen was obtained with the uterus at time of operation for carcinoma of the cervix. The corpus luteum was cystic, 2 to 4 cm. in diameter, and was filled with a clear, yellowish fluid. The convoluted border was yellowish gray, and 2 to 3 mm. thick.


The theca interna is very conspicuous in this specimen, being several cell layers thick around the entire granulosa lutein layer (fig. 21, pl. 4). The nuclei stain uniformly and present the appearance of actively functioning cells. For the first time since the stages of the mature follicle and the 16-day corpus luteum of the nonpregnant menstrual cycle, a number of small, irregular stellate cells with wrinkled hyperchromatic nuclei and homogeneous eosinophilic cytoplasm are seen in the theca lutein layer. Except for somewhat smaller size, these cells are identical in all respects with the K cells noted in routine haematoxylin and eosin sections of the younger corpora lutea. The sudden reappearance of these cells in the theca lutein layer is unexplained. There is no evidence of transformation or degeneration of theca lutein cells to these forms. The capillaries and sinusoids of the theca lutein layer are widely dilated.


The granulosa lutein layer at this stage appears highly disorganized. Cell boundaries are quite indistinct. The individual granulosa lutein cells show marked variation in the stainability of their nuclei and cytoplasm. There are a large number of patent capillaries in the granulosa lutein layer. Around each of these capillaries, proliferation of perivascular connective-tissue elements is seen. There is a definite increase in the reticular network that at this stage encircles nearly every granulosa lutein cell. The central coagulum is well organized, and large, dilated vascular channels are present in this new connective tissue.


Very few K cells that may be regarded as active are seen, but large numbers of these cells in all stages of degeneration are present. Colloid is present in larger amounts, and as a corollary the number of large, empty vacuoles is also increased over the preceding stage.


Most of the theca lutein and granulosa lutein cells contain very fine lipid droplets in large quantities (fig. 22, pl. An occasional theca lutein cell contains a few medium-sized lipid droplets. A significant number of the granulosa lutein cells contain large amounts of lipid in large, coarse droplets, indicating fatty degeneration. The K cells are still quite prominent and markedly sudanophilic, but they appear frayed and fibrillar. They are definitely not so numerous or attenuated as in figure 19. Many large, smooth sudanophilie globules are present. These match in number, size, shape, and location the colloid droplets seen in haematoxylin and eosin sections.


Of particular interest is the appearance of large numbers of these characteristic sudanophilic K cells scattered among the cells of the theca lutein layer. Both examples of 26-day pregnancy show them, and although carefully searched for, they were not seen in any other specimen. These cells appear to be very similar to the K cells observed in the early stages of development of the corpus luteum, and do not present the frayed, fibrillar appearance noted in the K cells in the granulosa lutein layer of this same specimen, facts which suggest that these cells represent younger forms.


The theca interna is uniformly devoid of alkaline phosphatase (Hg. 23, pl. 4), although most of the granulosa lutein cells contain moderate amounts of the enzyme. It is diflicult to recognize K cells in this preparation. The endothelium of blood vessels in both layers contains alkaline phosphatase.

A 28- to 35-Day Pregnancy, S48-4854

On gross examination the corpus luteum measured 2.3 by 2.0 cm. in its greatest diameters. The convoluted border was grayish yellow, measuring 2 to 3 mm. in thickness. The central coagulum was well organized.


The theca lutein layer is still very prominent, although not so conspicuous as in the preceding specimen. Many of the nuclei are dense and irregularly pycnotic. The cytoplasm of most of the cells is highly vacuolated (fig. 24, pl. 5). The vascular sinusoids of the theca interna are almost completely collapsed.


The granulosa lutein layer presents essentially the same picture as that of the preceding specimen in haematoxylin and eosin preparations. Figure 25, plate 5, is a photomicrograph of this same section, showing a clearly defined colloid globule. Other K cells in various stages of degeneration are present in this section.


The Sudan black preparations show large numbers of very fine peripherally distributed lipid granules in both theca lutein and granulosa lutein cells (fig. 26, pl. 5). Occasional cells undergong fatty degeneration are seen in both layers. The K cells are still quite conspicuous in the granulosa lutein layer, and all show a line dusting of sudanophilic droplets in their cytoplasm. A few small, uniformly sudanophilic cells similar to those described in the previous specimen are seen in the theca lutein layer, but these are not sufficiently conspicuous to photograph. A number of large, oily sudanophilic droplets resting in vacuoles may be noted among the granulosa cells. It is believed that this is the colloid mentioned earlier.


The alkaline phosphatase preparations are particularly striking. A number of the cells of the theca lutein layer contain large amounts of the enzyme, but most of the theca cells are totally devoid of it. On the other hand, the granulosa lutein layer contains a very high concentration of the enzyme, which is so diffusely distributed that it is impossible to separate the granulosa lutein cells from the K cells. Numerous large vacuoles, representing the site of colloid deposits that have been lost or dissolved in the process of preparation, are evident in this section as in figures 20 and 23.

A 4- to 4.5-Month Pregnancy, S48-2624

This specimen was obtained incidentally to a total hysterectomy performed during the 5th month of pregnancy because of carcinoma of the cervix. The corpus luteum measured 2 by 1.5 cm. The stigma was depressed and well healed. The periphery of the corpus luteum presented a yellowish, fatlike appearance upon the cut surface. Several small gelatinous areas were seen between the convolutions. There was a small hemorrhage, 2 by 5 cm., in the exact center of the gland.


This corpus luteum is fairly well preserved, as is indicated by the retention of many of the normal cellular relationships, particularly in the granulosa lutein layer. A striking feature, however, is that the theca lutein layer is represented by only a few scattered pycnotic nuclei (fig. 27, pl. 5). Nearly all the granulosa lutein cells are undergoing atrophy, and large, gaping vacuoles are scattered throughout the granulosa lutein layer. A number of colloid deposits are visible. The lutein layer is almost completely avascular. Connective-tissue organization is marked.


As in the haematoxylin and eosin preparations, the theca lutein layer is not evident in Sudan black treated sections. The granulosa lutein cells are uniformly devoid of lipid (fig. 28, pl. 5). An occasional K cell stands out conspicuously by virtue of its intense and uniform sudanophilia. Most of the K cells, however, appear only as “shadow forms,” having lost most of their sudanophilia. Though it is not evident in the photomicrograph, the fibrillar substructure of these cells is very conspicuous at higher magnification. Alkaline phosphatase is no longer demonstrable in the majority of granulosa lutein cells. A few cells along the outer margin of the lutein layer contain high concentrations of the enzyme (fig. 29, pl. 5). These cells occupy the same locus as the sudanophilic K cells mentioned above. It is of particular interest that the endothelium of blood vessels of both layers no longer contains demonstrable alkaline phosphatase.

Observations on Corpora Lutea associated with Abnormal Ova

In view of the fact that our material included 13 corpora lutea associated with ova that were adjudged to be abnormal on the basis of criteria stated earlier, it was decided to study these specimens closely to determine whether any relation between anatomical integrity of the corpus luteum and the condition of the ovum could be detected. These abnormal ova have been the subject of separate communications by Hertig and Rock (1944, 1949!), 1950).


Despite the fact that it is difficult to judge accurately the age of these ova, because of abnormalities of blastomeres, trophoblast, chorionic cavity, or germ disk, a reasonable estimate of age can be made from the appearance of the endometrium and from the history.

CORPORA I_.U’l'I-IA ASSOCIATED wrri-1 .A13:~.'-orm.-xi. FREE-LYING Ova

The corpora lutea associated with S43-I372 (Carnegie no. 8190), a 9—cell, 31/2" to 4-day free-lying segmenting ovum, and S46-3332 (Carnegie no. 8450), an 8-cell egg, are in no way different from the corpora lutea of the 17th and 18th days of the normal nonpregnant menstrual cycle.

A 5-Cell Egg, I9-Day Corpus Lute:-:m, S48-3948

The corpus luteum measured 2.0 by 1.8 cm. in its greatest diameters. The convoluted borders were pinkish gray, with a maximum thickness of 2 mm. The coagulum was pearly gray, and gelatinous in consistency.

The ovum consisted of five abnormal blastomeres, several of which were multinucleated and showed

other evidences of degeneration and retardation of development.

The corpus luteum differs in no respect from a 19-day corpus luteum of the normal nonpregnant menstrual cycle. The theca interna is quite prominent, several cell layers in thickness over the crests of the granulosa. The cytoplasm of the theca interna cells is markedly vacuolated. The granulosa lutein cells are plump and polyhedral, and only a few of them show peripheral vacuolization. A number of small capillaries and endothelial sprouts are seen in the granulosa lutein layer, but these capillaries are not dilated. K cells are not conspicuous in the routine haematoxylin and eosin sections.

Most theca lutein cells contain fine lipid droplets, but many cells contain only rnedium-sized to coarse lipid granules. Nearly all the granulosa lutein cells contain large amounts of fine peripherally distributed lipid. An occasional granulosa cell contains mediumsized lipid droplets. The K cells are not particularly prominent, showing only a moderate degree of uniform slate-gray sudanophilia.

Alkaline phosphatase is restricted to a few scattered clumps of cells of the theca lutein layer and the endothelium of blood vessels in both layers.

CORPORA LUTEA Assocuman WITH ABNoR:\r;tL Ova w1TH ADEQUATE TROPHOBLAST

An 8-Day Ozmm, Carnegie No. 8370, S46-676

The ovum is the youngest implanted embryo of this series of abnormal ova. The chorionic cavity is absent. Although ectoderm is present in the germ disk, there is no endoderm. The trophoblast is ade68 CORPUS LUTEUM OF HUMAN PREGNANCY

quate but poorly organized, that is, laminated instead of being concentrically arranged with the syncytiotrophoblast surrounding the cytotrophoblast.

The corpus luteum appears to be normal in all respects. The theca interna is prominent. Both layers are well vascularized. The granulosa lutein cells show a moderate degree of peripheral vacuolization. K cells are numerous and appear to be normally active. No evidence of colloid degeneration is seen.

A 10- to 11-Day Ouzrm, Carrzegie N0. 7770, S40-749

This ovum is not markedly defective. The trophoblast is moderately hypoplastic and the germ disk is maloriented.

The theca interna appears to be normal in all respects and consistent with that of a normal 10- or 11-day pregnancy. The granulosa lutein layer is moderately well vascularized. K cells are relatively few, but appear to be normal in all respects. There is, however, great variation in size and stainability of the nuclei of the granulosa lutein cells.

An Ir-Day Ozmm, Carnegie N 0. 8299, 545-1220

The trophoblast appears to be normal although somewhat poorly organized. The germ disk is markedly maloriented.

The corpus luteum is very large and consists of a large cystic cavity enclosed by a thin rim of lutein tissue. Both granulosa and theca lutein layers are moderately well vascularized and appear to be functionally active. Most of the K cells show little evidence of colloid degeneration. This appears to be a very active corpus luteum (fig. 30, pl. 6).

Carnegie No. 7850, 540-2699

This ovum is associated with an endometrium that evidences early decidual reaction. Except for moderate hypoplasia of the trophoblast, the ovum appears to be fairly good.

The theca interna is prominent, many cell layers thick, and appears to be functionally active. Peripheral vacuolization of the granulosa lutein cells is not so marked as in chronologically similar corpora lutea associated with normal pregnancy. Mitoses are to be noted in both layers of the corpus luteum. K cells are quite numerous, but are irregularly distributed. No evidence of colloid degeneration is to be noted anywhere in the section.

CORPORA Luri-:.-i ASSOCIATED wrrn ABNORMAL, SHaLLowi.v I.\tPL.~xN*rEn Ova

Ar: 11- to I2-Day Ourmz, Carnegie No. 8000, S42-217

The ovum is very shallowly implanted, although all the elements appear to be normal.

The theca lutein layer appears to be normal and contains several immature K cells. The granulosa lutein layer is moderately well vascularized, and although some lutein cells show early signs of atrophy, the majority appear to be functionally active. Distinctive K cells are rare, but little colloid degeneration is seen.

A I21/3-Day Oz/um, Carnegie No. 290, 844-2785

Although this ovum is superficially implanted, it shows evidence of early villus formation. The germ disk has undergone a curious buckling and is still attached to the trophoblast. The latter is irregularly developed, poorest at the implantation pole and, paradoxically, good elsewhere.

The corpus luteum is in no way markedly different from that associated with Carnegie no. 8000.

A Conpus LU'n~:U.\t Assoenvrsn w1TH .»'tN OVUM CoNSISTING or Si'Nev'r1oTRopi-ioBL.-xsT ONLY

A 12-Day Oz/um, Cczrnegic No. 8329, S45-1809

This embryo is markedly abnormal in that there is no cytotrophoblast, no segmentation cavity, and no germ disk, but as a mass of syncytiotrophohlast it is fairly large.

Both lutein layers appear to be well vascularized and functionally active. K cells are numerous in certain parts of the section, but almost completely lacking in other areas. There is, however, no evidence

of colloid degeneration of any of the K cells (fig. 31, pl. 6).

Coupons. Lure.-x Assoel.-cren wrrt-I OVA SHOWING HvPoi>1..»xs'r1e TROI’liUBL.-\S'l‘

Carncgz'e No. 7771, S4079!

This markedly abnormal ovum is associated with a 27-day late-secretory endometrium. The ovum is a polypoid structure; both elements of the trophoblast are present, but poor in quality and organization. The germ disk is totally lacking.

The corpus luteum is poorly vascularized. Both theca lutein and granulosa lutein cells appear to be moderately active, but not so much so as one would find in a normal pregnancy of this stage of development. A very striking feature is the complete colloid degeneration of all the K cells. Large amounts of colloid are present in all parts of the section, and no functional K cells are seen (fig. 32, pl. 6).

Carnegie N0. 7800, 540-1327

This ovum is associated with an endometrium that shows early decidual reaction. The chorion is irregularly deficient, with marked hypoplasia of the trophoblast. To judge from the development of the embryo, the chorion should have early villi.

The theca lutein layer appears to be normal and functioning. The granulosa lutein layer is well vascularized. There is marked variation in size, regularity, and stainability of the nuclei of the granulosa lutein cells. The majority of the K cells have undergone more or less complete colloid degeneration, although this feature is not so conspicuous as in Carnegie no. 7771.

About 3 Mont/is Pregnancy, Blig/ztcd Ouum, S48-824

This specimen was obtained with the corresponding left tube, left ovary, and uterus. Pregnancy was interrupted because of impending cardiac failure. The placenta was found to be immature and associated with a blighted ovum of about 3 months menstrual age. The cystic corpus luteum measured 1.5‘ cm. in diameter and contained straw-colored fluid. The lutein border was grayish yellow, and about 2 mm. thick.


Haematoxylin and eosin sections (fig. 33, pl. 6) show a corpus luteum that has lost all its distinctive morphologic features. The theca lutein layer is markedly reduced in bulk. Most of the nuclei are densely pycnotic. The granulosa lutein layer has lost all evidence of functional activity. Not a single active lutein cell can be found. In many, the nuclei are dense and pycnotic, and in others the nuclei have completely lost their capacity for staining. A large number of colloid-containing vacuoles are present. Cell boundaries are totally obliterated. Connectivetissue invasion of both layers is marked. The capillaries of the granulosa lutein layer are collapsed.


Only an occasional cell of the theca lutein layer contains alkaline phosphatase. Nearly every cell in the granulosa lutein layer contains some of the enzyme, but not in significant amounts.


Inasmuch as the ovum was found to be blighted, and the placenta was immature, it is highly probable that this specimen does not represent a normal 3 months pregnancy. It is included to demonstrate that the degenerative changes in the Corpus luteum may parallel trophoblastic degeneration.

Unfortunately, no formalin-fixed material was available at the time this specimen came to our attention, and lipid studies could not be made.

Notes on the K Cells

Although this study was undertaken to outline the morphological changes that take place in the corpus luteum during early pregnancy, it became apparent quite early in the course of the investigation that the K cells described in all specimens discussed bear more than a casual relation to the cyclical activity of the gland, in the nonpregnant cycle as well as in early pregnancy.


These cells are conspicuous in routine sections because of their homogeneous, highly cosinophilic cytoplasm. It is also to be noted, however, that in sections treated with basic dyes, their cytoplasm is more markedly basophilic than that of neighboring lutein cells. This suggests strong acid properties of the protoplasm, a reaction characteristic of phospholipid. As previously stated, these cells are very striking in sections treated with Sudan black. They are uniformly sudanophilic, in contrast with the granular sudanophilia of the theca lutein and granulosa lutein cells. The sudanophilic material is soluble with difficulty in cold alcohol or alcohol-acetone mixtures. Traces of sudanophilia can be detected in these cells after 24 hours’ treatment of the section in lipid solvents before staining. By contrast, the granular deposits of lipid in theca lutein and granulosa lutein cells are completely dissolved by similarly pretreating the sections for 1/3 to 1 hour before staining with Sudan black. The low solubility of the sudanophilic substance in these K cells is characteristic of phospholipid.


Because of these indications of high phospholipid content in the K cells, the technique for the demonstration of phospholipid in tissues (Baker, 1946) was employed in two selected corpora lutea. Figure 34, plate 7, is a photomicrograph of a K cell, at a magnification of 750, in a :20-day corpus luteum of the normal menstrual cycle, showing a high concentration of phospholipid. The phospholipid is uniformly distributed in these cells, as contrasted with the lutein cells of both layers, where phospholipid appears as peripherally distributed granules. It was observed incidentally that in the developing follicle phospholipicl is restricted to the theca interna layer. Pretreatment of parallel sections with pyridine completely removed all traces of phospholipid from the gland.


All the specimens discussed in this paper were subjected to the tests for presumptive ketosteroids employed so effectively by Dempsey and Bassett (1943) and by McKay and Robinson (1947). Our findings in the developing follicle and corpus luteum of the nonpregnant cycle agree substantially with those of McKay and Robinson. It was noted, however, in the course of these studies that the K cells were reactive with the Schiff, phenylhydrazine, and LiebermanBurchardt reactions. This reactivity is manifested as a uniform, homogeneous color reaction, in contrast with the granular reactivity of theca lutein cells described by McKay and Robinson. As with Sudan black, this reactive material was more dillicult to dissolve in lipid solvents than that of the theca lutein cells. Observations of autofluorescence under ultraviolet light were equivocal. It is noteworthy, however, that birefringent crystals cannot be observed in these K cells under the polarizing microscope. In only one specimen, the 16-day normal pregnancy (Carnegie no. 8602), was a suggestion of crystalline birefringence seen. Up to this point, all the reactions of the K cells pointed to ketosteroid substances, but failure to demonstrate birefringent crystals in them seemed to rule out that possibility. It has subsequently been pointed out to us by Seligman (personal communication) that all the evidence presented above points to the fact that the high phospholipid content of these cells may prevent the crystallization of ketosteroid necessary for development of birefringence in plane-polarized light.


Very recently Seligman and Ashbel (I949) developed a technique for the demonstration of ketonic lipids. Figures 35 and 36, plate 7, are photomicrographs of sections treated by this technique. It will be noted that reactive material is concentrated in these cells. Seligman and Ashbel have observed that the corpora lutea of animals do not react to the tests for ketosteroid unless they are fixed in formalin, which unmasks the active carbonyl group. To rule out the possibility that the reactive material in these cells might be an aldehydic group, produced by hydrolysis of plasmogens, parallel sections were treated by a new technique for the demonstration of free aldehyde groups (Seligman and Ashbel, unpublished data). No free aldehydic groups could be demonstrated. Thus, all the evidence seems to indicate that these K cells represent the locus of high concentration of ketosteroid. Their reactivity as measured by these special histochemical techniques parallels closely the changes observed in sudanophilia during various stages of development of the corpus luteum.


As regards the question of origin and function of the K cells, it must be borne in mind that they could represent undifferentiated elements of the reticuloendothelial system, for example wandering macrophages, angioblasts, or young fibroblasts. It is possible that these cells arc histiocytic, and that the intense localization of ketonic lipid noted in them represents merely the phagocytosis of excess ketonic lipid. It is planned to perfuse a human ovary containing an active corpus luteum with a vital dye to determine whether these cells have the capacity to phagocytose foreign matter, utilizing the technique of ‘Nerthessen (1949). In the absence of further and more definitive studies, we believe that all the evidence presented regarding the peculiar characteristics of these cells points to their being a distinct cell line, intimately related in some manner to the function of the corpus luteum in the production or utilization of ketonic lipid, that is, ketosteroid compounds.

Discussion

Our observations of the corpus luteum of the nonpregnant cycle parallel substantially the classic description of Meyer (1911). Observations of vascular changes, coupled with information that can be deduced from histochemical procedures, lead us to agree with Brewer (1942) that the corpus luteum reaches its maximum activity on or about the 9th day after ovulation. ‘Ne have assumed the following criteria as evidence of functional activity of the corpus luteum: widely dilated capillaries in both theca and granulosa lutein layers, fine peripherally distributed sudanophilic substances in the lutein cells, demonstrable alkaline phosphatase in the cytoplasm, and ketonic lipid demonstrable by the techniques described above.


It has been generally held that the ovary is responsible for at least two hormones, both of which have experimentally proved specific actions in the menstrual cycle. Dempsey and Bassett (1943) working with rats, and McKay and Robinson (1947) in a study of human material, found that reactive material, presumptively ketosteroid, was localized exclusively in the theca interna of the developing follicle and appeared in insignificant amounts in the granulosa lutein cells during the active stages of the corpus luteum. Inasmuch as they could not demonstrate ketosteroid in significant amounts in the granulosa lutein layer, they suggested that the theca interna is probably responsible for the production of both progesterone and estrogenic substances. Our observations suggest that a ketosteroid substance is intensely localized in a specific line of cells having their origin in the theca interim and making their way into the granulosa lutein layer. It is possible that the hormone production of these cells in the unruptured follicle is responsible for the suggestive progestational changes that take place in the uterus of experimental animals at or shortly before ovulation (Reynolds and Friedman, I93o; Astwood, 1939).


We have no evidence that these cells are specifically concerned with the production of progesterone rather than estrogen. Nevertheless, the temptation to infer that such a relationship exists is strong. There is no valid reason why a given cell cannot produce more than one hormone, as those of the pituitary gland apparently do. As has been observed, these K cells show the greatest evidence of functional activity dur ing the stages when maintenance of the progestational state of the uterine endometrium is essential. In the normal menstrual cycle, if a fertilized ovum has not become implanted in the endometrium by 6 or 7 days after ovulation, that cycle ends in menstruation (Rock and Hertig, 1948). In such an event, the need for maintenance of the progestational type of endometrium no longer exists and the corpus luteum soon begins to regress. On the other hand, if pregnancy is superimposed, the earliest evidence of sustained and accelerated functional activity is seen in the recrudescence of activity in the K cells. It has been generally agreed that pregnancy will continue uninterrupted even though the corpus luteum is removed, when the placenta is producing suflicient progesterone to maintain the decidua. The exact time at which this transfer of function from corpus luteum to placenta takes place is not so well agreed upon. Dr. G. van S. Smith (unpublished data) states that the corpus luteum is essential through the 7th to the 8th, and possibly the 9th, week of pregnancy. No doubt there is considerable overlapping of hormone production by corpus luteum and placenta. Our observations suggest that the corpus luteum ceases to produce hormone completely by the 4th month of pregnancy, and there is evidence that functional activity decreases rather sharply between the 7th and 12th weeks of gestation.


Our studies indicate that the theca interna contributes substantially to the granulosa lutein layer in the human corpus luteum. However, the theca lutein and granulosa lutein cells described by all workers remain separate and distinct entities. The elements contributed to the granulosa lutein layer by the theca lutein layer are the K cells to which so much space has been given in this discussion.


These K cells in the granulosa lutein layer of the human corpus luteum have been observed by many earlier workers, as cited by Gillman and Stein (1941). These latter authors have interpreted them as representing different phases of functional activity of the granulosa lutein cells. It is highly probable that earlier work failed to elicit the true nature of these cells for three reasons: First, they had not been observed in the theca interna of the developing follicle; secondly, there had been no observation of migration of cellular elements from the theca interna into the granulosa lutein layer; and, thirdly, lipid stains such as Sudan black, which is capable of staining the phospholipid so characteristic of these cells, were not available.


The specific colloid degeneration which is peculiar to these K cells is interpreted as further evidence that they represent a distinct cell type. The fact that this colloid has the same histochemical properties as the K cells suggests that the colloid represents stored secretory products. Although colloid deposits are visible in old corpora lutea of the nonpregnant cycle, they are much more conspicuous in the degenerating corpus luteum of pregnancy, a fact which suggests both an increase in the number of cells producing the precursor of this product and an increased concentration of precolloid ketosteroid-phospholipid complexes within the cell.


As observed by Corner (19.18), in the human ovary alkaline phosphatase is restricted to the theca interna of the developing follicle and disappears from the theca lutein cells by the 4th or 5th post-ovulatory day. In contrast with Corner’s findings, we observed alkaline phosphatase in a few granulosa lutein cells, but this becomes evident only as the gland approaches the peak of functional activity, that is, at or about 8 days after ovulation. As stated earlier, we have observed alkaline phosphatase in the K cells as early as 6 days after rupture of the follicle. V-Vhen pregnancy occurs, alkaline phosphatase appears in increasingly higher concentrations in the K cells, and in gradually increasing amounts in the true granulosa lutein cells as the progress of pregnancy makes additional demands on the corpus luteum. There is some recrudescence of alkaline phosphatase in the theca lutein cells, most marked at 6 or 7 weeks menstrual age. The activity of the theca lutein layer during pregnancy, as measured by the concentration of this particular enzyme, is insignificant when compared with that of the granulosa lutein layer and the K cells.


The theca lutein layer undergoes marked hypertrophy during early pregnancy, attaining its maximum development about the 26th day of pregnancy, dating from ovulation. Early in pregnancy, the coarse lipid deposits characteristic of the menstrual corpus luteum are completely replaced by fine, granular lipid deposits which are indicative of functional activity. After the 26th day of pregnancy the theca lutein layer becomes less and less prominent, until at 4 months no trace can be found except a few small pycnotic nuclei. That the theca interna may continue to supply K cells is suggested by the fact that recrudescence of K cells in the theca lutein layer is observed in both the corpora lutea associated with 26-day pregnancies.


Another very important question that remains to be elucidated is the role of the granulosa lutein cell. The fact that alkaline phosphatase concentration in the granulosa lutein cell increases during that period of pregnancy when greatest demands are being made upon the gland suggests that the granulosa lutein Cell plays more than a passive role. It is possible that the short 3-hour period of incubation in the glycerophosphate medium employed in this study was insufficient to reveal low concentrations of alkaline phosphatase in the granulosa lutein cells of the nonpregnant corpus luteum. Here, again, it is a tempting suggestion that there may be a direct relation between alkaline phosphatase in the granulosa lutein cell and the demonstrable phospholipid-ketosteroid matrix of the K cells.


As regards the corpora lutea associated with abnormal ova, it is interesting to note that the corpora lutea associated with preimplantation ova are normal in all respects and comparable with those of the normal menstrual cycle or normal pregnancy of identical chronological age.


Careful examination of the corpora lutea associated with implanted but abnormal ova discloses that there is an almost direct relation between the amount of trophoblast present and the integrity of the corpus luteum. In those ova which are almost completely devoid of trophoblast (Carnegie nos. 7771, 7800), the corpus luteum is poorly vascularizcd and does not manifest the heightened functional activity expected at this stage of development. The most conspicuous feature of these corpora lutea, however, is the total and uniform colloid degeneration of all K cells.


On the other hand, Carnegie no. 8329 is a markedly abnormal ovum consisting only of syncytiotrophoblast. K cells are numerous in this corpus luteum, and no evidence of colloid degeneration is to be noted. The specimens associated with abnormal ova showing only moderate hypoplasia of the trophoblast appear to be good but not perfect corpora lutea; that is, peripheral vacuolization and vascularity are moderately deficient.

These observations suggest that a normal trophoblast is essential to the maintenance of the corpus luteum, the functional integrity of which, in turn, is responsible for maintaining normal decidua during the early weeks of pregnancy.

Summary and Conclusions

1. The morphological and histochemical changes have been studied in a total of 89 human corpora lutea. Forty-eight of these represent corpora lutea of the normal menstrual cycle, every day from ovulation to menstruation being represented. Twenty—eight corpora lutea of normal pregnancies ranging from the 2-cell egg to 4%, months, and 13 corpora lutea associated with abnormal ova were studied. An attempt has been made to correlate the changes in histochemical reactivity in the various components of the corpus luteum with the anatomical evidences of function.

2. As the mature Graafian follicle nears ovulation, a number of distinctive cells, not previously described, become conspicuous. At or shortly after the time of ovulation, these cells appear in large numbers in the granulosa lutein layer, attaining that position by their own motility or being carried in as the theca interna and its accompanying blood vessels invaginate into the collapsed membrana granulosa.

3. Evidence is presented that these cells constitute a distinct cell type and represent the site of intense localization of ketonic lipid, if not the site of production or utilization of ketonic lipids or steroids.

4. Until the time of implantation of the ovum in the endometrium, no difference can be noted between chronologically similar corpora lutea of the normal menstrual cycle and those associated with normal or abnormal ova.

5. After implantation, at or about 6 or 7 days after ovulation, the corpus luteum does not undergo regression but is stimulated to increasingly higher levels of functional activity until 6 weeks of menstrual age or later, after which the function of the corpus luteum is gradually taken over by the placenta.

6. It is quite apparent that when the implanted ovum is deficient in trophoblastic development, the corpus luteum undergoes early regression. The most striking feature of this failure of the corpus luteum is the uniform colloid degeneration of the K cells. The worse the ovum as regards the development of the trophoblast, the more complete is the colloid degeneration of the K cells in the associated corpus luteum.

Literature Cited

Asor.1.1., S. A. 1928. The growth and function of the corpus luteum. Physiol. Rev., vol. 8, pp. 313-341.

As:-1m«;1., R., and A. M. St-:1.1o.\1.-m. 1949. A new reagent for the histochemical demonstration of active carbonyl groups. A new method for staining ketonic steroids. Endocrinology, vol. 44, pp. 565-583.

Asrwoon, E. B. 1939. Changes in the weight and water content of the uterus of the normal adult rat. Amer. ]0ur. Physiol., vol. 126, pp. 162-170.

BAKER, I. R. 1946. The histochemical recognition of lipine. Quart. Iour. Micr. Sci., vol. 87, pp. 441-470.

BENNETT, H. S. 1940. The life history and secretion of the cells of the adrenal cortex of the cat. Amer. Iour. Anat., vol. 67, pp. 151-22 .

BREWER, J. I. 1942. Studies of the human corpus luteum. Evidence for the early onset of regression of the corpus luteum of menstruation. Amer. Iour. Obstet. and Gynecol., vol. 44, pp. 1048-1059.

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Conxra, G. \V. 1915. The corpus luteum of pregnancy, as it is in swine. Carnegie Inst. VVash. Pub. 222, Contrib. to Embryol., vol. 2, pp. 69-94.

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DALTON, A. L, E. R. M1Tct1ELL, B. F. Joints, and V. B. Perms. 1944. Changes in adrenal glands of rats following exposure to lowered oxygen tension. Iour. Natl. Cancer Inst., vol. 4, pp. 527-536.

D1—:1.es1'ar_, M. 1910. Recherches sur le follicule de Graaf et le corps jaune de la vache. Iour. de l‘anat. et de la physiol. (Paris), vol. 46, pp. 286-309.

D1-:1~.11>s1-:v, E. ‘W. 1948. The chemical cytology of endocrine glands. Recent Prog. Hormone Res., vol. 3, pp. 127-157.

and D. L. B.1ss1»:'r1'. 1943. Observations on the fluorescence, birefringence and histochemistry of the rat ovary during the reproductive cycle. Endocrinology, vol. 33, pp. 334-401

GILLMAN, 1., and H. B. STEIN. 1941. The human corpus luteum of pregnancy. Surg., Gynecol., and Obstet., vol. 73: PP- I39‘149 Gomoar, G. 1941. The distribution of phosphatase in normal organs and tissues. Iour. Cell. and Comp. Physiol., vol. 17, pp- 7I-83

GRAFFLIN, A. L. 1939. The thyroid and parathyroid glands of the Barasingha deer, with particular reference to autolluorescence, fat and pigment. Iour. Morphol., vol. 65, pp- 297-32!


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