Superoxide dismutase expression in the human corpus luteum during the menstrual cycle and in early pregnancy

To investigate the possible role of the superoxide radical and its scavenging system in the human corpus luteum, superoxide dismutase (SOD) values and lipid peroxide concentrations were analysed in the corpora lutea during the menstrual cycle and in early pregnancy. Copper-zinc SOD (Cu,Zn-SOD) activities increased from the early to mid-luteal phase, and gradually decreased thereafter and were the lowest in the regression phase. In pregnant corpus luteum, Cu,Zn-SOD activities were significantly higher than those in the mid-luteal phase. In contrast, manganese SOD (Mn-SOD) activities were low in the mid-luteal phase and increased toward the regression phase. Changes in mRNA expression of both types of SOD were similar to changes in their activities. Lipid peroxide concentrations were the highest in the regression phase whereas they were remarkably low in pregnant corpus luteum. The effects of human chorionic gonadotrophin (HCG) on luteal SOD were studied in vitro. HCG significantly increased Cu,Zn-SOD expression in mid-luteal phase corpora lutea, but not in late luteal phase corpora lutea. In conclusion, the present study suggests that the superoxide radical and its scavenging system, especially Cu,Zn-SOD, play important roles in the regulation of human luteal function. The stimulation of luteal Cu, Zn-SOD expression by HCG may be important in maintaining luteal cell integrity when pregnancy occurs.     

Ultrastructure of the corpus luteum of the white-tailed deer during pregnancy

The fine structure of corpora lutea from 14 white-tailed deer was studied from early through near-term pregnancy. The corpora contained both thecal and granulosal lutein cells. The small, elongate and cylindrical thecal lutein cells contained many lipid droplets, juxtanuclear Golgi elements, abundant agranular endoplasmic reticulum, and other cytoplasmic organelles. These cells were observed throughout pregnancy. Some of the thecal lutein cells became modified during early to midpregnancy. The modified thecal lutein cells possessed many lipid droplets, several lysosomes, packets of PAS-positive glycogen granules, and numerous small membranous whorls of agranular endoplasmic reticulum. The granulosal lutein cells had highly folded and/or ruffled plasma membranes, abundant tortuous tubular and cisternal agranular endoplasmic reticulum, numerous rod-shaped to round mitochondria with tubular and lamellar cristae, Golgi elements, and a few packets of granular endoplasmic reticulum. Many granulosal lutein cells became modified during mid- to near-term pregnancy by the addition of numerous small membrane-bound osmiophilic droplets and variable numbers of large nonmembrane-bound lipid droplets. The granulosal and thecal lutein cells appeared more active in steroid biosynthesis than the modified granulosal and thecal lutein cells.     

Angiogenesis in the human corpus luteum: simulated early pregnancy by HCG treatment is associated with both angiogenesis and vessel stabilization.

BACKGROUND: This study examined changes in the luteal vasculature throughout the menstrual cycle and during simulated pregnancy with human chorionic gonadotrophin (HCG) in the human. METHODS: Endothelial cell and pericyte area were assessed by quantitative immunocytochemistry for CD34 and alpha-smooth muscle actin respectively, taking into consideration the dynamics of lutein cell hypertrophy and atrophy throughout the cycle and after HCG treatment. Endothelial cell proliferation was detected by Ki-67/CD34 dual staining and a proliferation index was obtained. The molecular regulation of angiogenesis was studied by examining changes in vascular endothelial growth factor (VEGF) immunostaining. RESULTS: The early luteal phase is associated with intense angiogenesis, as indicated by high endothelial cell proliferation, and by the mid-luteal phase a mature vasculature was apparent, as shown by maximal endothelial cell and pericyte areas. During the late luteal phase, decreased endothelial proliferation, endothelial cell and pericyte area indicated vascular regression. HCG treatment induced a second burst of total and endothelial cell proliferation and a concomitant increase in endothelial cell and pericyte areas. VEGF protein was expressed throughout the luteal phase and a significant increase was found after HCG treatment. CONCLUSION: Luteal rescue with HCG is associated with a second wave of angiogenesis and vascular stabilization.     

Histology of the human corpus luteum of early and late pregnancy.

The aim of this investigation has been to examine which histological parameters are of value in the age determination of the corpus luteum of pregnancy. The material comprises representative ovarian biopsies from 16 women from the 10th through 42nd week of gestation. The following histological parameters have been found useful for the age determination of the corpus luteum: 1) the size of the granulosa cells; 2) the number of K-cells, vacuoles and colloid inclusions in the granulosa cell layer; 3) the size of the theca interna cells; (4) the size of the vessels in the theca interna. Futhermore, the differentiation of the corpus luteum of pregnancy from the corpus luteum of menstruation is described, and some indications for a histological age determination of the corpus luteum of pregnancy are mentioned.     

The fine structure of the human corpus luteum of early pregnancy and during the progestational phase of the menstrual cycle

The fine structure of granulosa lutein cells and theca lutein cells of human corpora lutea of early pregnancy and during the progestational phase of the menstrual cycle (i.e., 1 day, 7 days and 9 days after estimated ovulation) is described. Granulosa lutein cells of early pregnancy are distinguished from theca lutein cells in having: (1) a more homogeneous, electron-lucent nuclear matrix, (2) enlarged pleomorphic mitochondria with irregularly-shaped, osmiophilic inclusions, (3) numerous isolated regions of the Golgi complex, (4) abundant whorls of granular and agranular endoplasmic reticulum, (5) a folded-membrane complex, (6) numerous bundles of 50 Å filaments and (7) patches of elongated microvilli bordering intercellular and intracellular canaliculi. Furthermore, granulosa lutein cells of early pregnancy are distinguished from granulosa lutein cells of corpora taken during the progestational stage of the menstrual cycle by the greater abundance of granular and agranular endoplasmic reticulum, the presence of concentric membranous whorls and large spherical mitochondria, increases in membrane-bound granules and a more extensive development of intracellular canaliculi. These differences are related to the high titers of serum gonadotrophin (s) during early gestation. The morphology of human corpora lutea is compared with steroidogenic tissues of other species and is correlated with the capacity of human corpora lutea to synthesize estrogens in addition to progestogens. Based upon morphological evidence, it is suggested that in addition to elaborating steriod hormones, corpora lutea may also secrete a proteinaceous product, perhaps relaxin.

1 This work was presented as a demonstration to the eighty-second annual session of the American Association of Anatomists in April, 1969. An abstract has been published (Anat. Rec., 163: 336, '69).

     

The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis,and the phagocytosis of luteal cells

Little information is available on the ultrastructure of macrophages in the corpus luteum or their importance in the regression of luteal tissue. In the present study, the fine structure of activated luteal macrophages during pregnancy and the postpartum period was examined by electron microscopy of guinea pig ovaries fixed by vascular perfusion. In these corpora lutea, macrophages can readily be distinguished from luteal cells. Activated macrophages typically display three prominent inclusions in their cytoplasm: (1) heterophagic vacuoles, (2) distinctive large dense inclusions, and (3) large and small electron-lucent vacuoles. In addition, they contain numerous smaller lysosome-like dense bodies. Activated macrophages in corpora lutea also characteristically show many surface protrusions, such as processes, folds or pseudopodia, which often occur in close contact with nearby luteal cells. Generally, nuclei of macrophages are irregular in shape and display a dense border of heterochromatin, thus differing from those of luteal cells. Macrophages seem to be most abundant in regressing corpora lutea, where they commonly display heterophagic vacuoles containing recognizable luteal cell fragments, evidence that these phagocytes ingest senescent luteal cells. The digestion of luteal cell components in heterophagic vacuoles presumably gives rise to the distinctive large dense inclusions typically seen in macrophages. The findings of this study indicate that macrophages play a central role in luteolysis by phagocytizing luteal cells or their remnants. They therefore appear to bring about the reduction in volume of the corpus luteum that occurs as this tissue regresses. These results taken together with those previously published (Paavola, 1978) further indicate that breakdown of the corpus luteum during postpartum luteolysis in guinea pigs involves both autophagy and heterophagy.     

Immunolocalization of glutaredoxin in the human corpus luteum.

Glutaredoxin (Grx) is a small protein with oxidoreductase activity which is involved in the cellular defence against oxidative stress. Corpus luteum (CL) regression has been related to the generation of reactive oxygen species (ROS). We have studied the presence of glutaredoxin in the human ovary during the ovulatory cycle using polyclonal antibodies developed against recombinant human Grx. Immunostaining was only detected between days 15 and 23 of the cycle and was localized exclusively in the corpus luteum. Grx-positive cells corresponded to granulosa-derived luteal cells (GLC) whereas the remaining luteal cell types were not immunostained. In general, Grx immunoreactivity was parallel to the functional activity of the CL. Most GLC were immunostained on days 15-16 of the cycle, whereas on days 17-19 immunoreaction was found mainly at the inner and outer aspects of the granulosa lutein layer (GLL). After this stage only isolated GLC showed Grx immunoreactivity and no reaction was found from day 23 of the cycle onward. In two CL of pregnancy that were also studied, isolated GLC showed Grx immunoreactivity. Loss of Grx immunoreactivity was coincident with the appearance of morphological signs of structural luteolysis, such as shrinkage of the GLL and the presence of apoptotic cells. These data suggest that Grx, as a cellular antioxidant, plays an important role in the mechanisms of human CL development.     

Immunolocalization of bcl-2 in the human corpus luteum

The mechanisms of luteal regression and rescue in women areunknown but forms of programmed cell death may be involved.The proto-oncogene bcl-2 is an important inhibitor of apoptosisbut has not previously been described in the human corpus luteum.Immunohistochemical localization of bcl-2 protein was investigatedin human corpora lutea obtained from women undergoing surgeryduring endocrine monitored menstrual cycles as well as fromwomen who had been treated with human chorionic gonadotrophin(HCG) to prolong the luteal phase. Bcl-2 was found to be localizedin granulosa-lutein, theca-lutein (as identified by co-localizationof P450_{17-hydroxylase}) and the endothelial cells around someblood vessels. Immunoblotting demonstrated the presence of asingle band of MW 26 kDa. There was no apparent change in eitherthe intensity of immunostaining or the histological localizationduring the normal luteal phase or following treatment with humanchorionic gonadotrophin. The product of the proto-oncogene bcl-2is present in the human corpus luteum. It is unlikely that bcl-2expression alone is responsible for prolongation of the lifespanof the corpus luteum in early pregnancy although it is possiblethat the action of the bcl-2 gene present is modified by changesin other members of the bcl-2 family. apoptosis/bcl-2/corpus luteum/granulosa/17-hydroxylase     

Extracellular matrix of the human cyclic corpus luteum

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV alpha1 and laminins alpha5, beta2 and gamma1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV alpha1 and laminins alpha2, alpha5, beta1 and beta2 localized in the interstitial matrix. Laminin alpha4 and beta1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin alpha1 and alpha3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.     

Alternative splicing of the human luteal LH receptor during luteolysis and maternal recognition of pregnancy

Deletion of exon 10 of the human LH receptor impairs LH but not hCG action. Other splice variants of the LH receptor impair both LH and hCG action in other species. We hypothesized that alternatively spliced LH receptors are involved in luteolysis and luteal rescue with hCG in women. mRNA was extracted from human luteinized granulosa cells and from corpora lutea from across the luteal phase and after luteal rescue in vivo with exogenous hCG. Splice variants were detected by RT-PCR using carefully designed primer pairs. Products were visualized on agarose gels, extracted, purified and sequenced. Three splice variants of the human LH receptor were detected and characterized. These demonstrate a region of multiple splicing between exons 8 and 11 of the receptor. A naturally occurring splice variant with exon 10 alone removed was not identified. There was no obvious change in the pattern of splice variants across the luteal phase in the presence or absence of hCG. These data do not support the hypothesis that qualitative changes in LH receptor splicing have a role in luteolysis or that a naturally occurring LH receptor lacking exon 10 has a role in maternal recognition of pregnancy.     

Cell cycle: Immunolocalization of bcl-2 in the human corpus luteum

The mechanisms of luteal regression and rescue in women areunknown but forms of programmed cell death may be involved.The proto-oncogene bcl-2 is an important inhibitor of apoptosisbut has not previously been described in the human corpus luteum.Immunohistochemical localization of bcl-2 protein was investigatedin human corpora lutea obtained from women undergoing surgeryduring endocrine monitored menstrual cycles as well as fromwomen who had been treated with human chorionic gonadotrophin(HCG) to prolong the luteal phase. Bcl-2 was found to be localizedin granulosa-lutein, theca-lutein (as identified by co-localizationof P450_{17-hydroxylase}) and the endothelial cells around someblood vessels. Immunoblotting demonstrated the presence of asingle band of MW 26 kDa. There was no apparent change in eitherthe intensity of immunostaining or the histological localizationduring the normal luteal phase or following treatment with humanchorionic gonadotrophin. The product of the proto-oncogene bcl-2is present in the human corpus luteum. It is unlikely that bcl-2expression alone is responsible for prolongation of the lifespanof the corpus luteum in early pregnancy although it is possiblethat the action of the bcl-2 gene present is modified by changesin other members of the bcl-2 family.     

Distribution of leukocyte subpopulations in the human corpus luteum.

Cytokines, as secreted products of leukocytes, have roles in many organs of the body via paracrine or autocrine mechanisms. In the present study, we demonstrate by immunocytochemistry the leukocytes present in the human corpus luteum in order to investigate further the relationship between leukocytes, cytokines and corpus luteum function. Ten intact corpora lutea were collected from female patients who had no apparent ovarian disease. The mean age of these patients was 37 years (range 23-55 years). Frozen and paraffin sections were subjected to analysis using monoclonal antibodies which were specific to leukocyte marker antigens. The results showed that there are macrophages, cells positive for leukocyte common antigen (LCA), T lymphocytes including T helper/inducer (T4) cells, T cytotoxic/suppressor (T8) cells and activated T (Ta) cells (interleukin-2 receptor-positive cells), monocytes and natural killer (NK) cells but not B lymphocytes present in the human corpus luteum. The distribution of the leukocytes present in the different parts of the corpus luteum was found to be in the order: theca-luteal area greater than loose connective tissue area greater than granulosa-luteal area. Macrophages and T lymphocyte subsets comprised the main components of the total leukocytes in the human corpus luteum. Ta cells were only localized in the loose connective tissue of the corpus luteum. In most cases, macrophages, LCA cells and T4 cells tended to be situated in a single cell layer on the edge of the theca-luteal area and surrounding the granulosa-luteal area. These results suggest that the leukocytes may act to a greater extent in the theca-luteal area than in the granulosa-luteal area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary results on the role of embryonic human chorionic gonadotrophin in corpus luteum rescue during early pregnancy and the relationship to abortion and ectopic pregnancy.

The precise mechanisms by which corpus luteum (CL) function is modulated during early pregnancy are not known. Evidence in failed pregnancies (ectopic, abortions), shows that factors other than human chorionic gonadotrophin (HCG) could be involved in its regulation. The objective of this study was to investigate the dynamics of beta-HCG, progesterone and oestradiol production in early pregnancy and its relation to embryonic quality and topographic localization. Plasma concentrations of progesterone, oestradiol and beta-HCG were studied between days +12 and +21 after an in-vitro fertilization (IVF) embryo transfer in 11 intrauterine pregnancies, 10 intrauterine abortions and seven tubal pregnancies. Tubal pregnancies and abortions were grouped according to doubling time (DT) of HCG. Results showed that oestradiol concentrations were apparently reduced in both ectopic pregnancies and abortions compared with normal pregnancies. The fall in oestradiol concentrations was seen in ectopic pregnancies with an abnormal DT for HCG and in all abortions. When the ectopic pregnancy had a normal DT, oestradiol and progesterone concentrations were normal. In abortions, the fall in oestradiol and progesterone concentrations was less influenced by the DT of HCG. These findings suggest that corpus luteum function depends on an adequate DT of HCG more than an absolute value, and with normal trophoblastic tissue the site of implantation does not affect CL function.     

Localization of prostaglandin endoperoxide synthase in the human corpus luteum

Prostaglandins have been implicated in both maintenance and luteolysis of the primate corpus luteum. Central to the production of prostaglandins is the enzyme prostaglandin endoperoxide synthase (PGHS). In the present study, we identified the cell types which contain PGHS in 44 human corpora lutea, using immunoperoxidase staining techniques. Intense granular staining was present in the cytoplasm of granulosa lutein cells of tissues obtained from the mid-luteal phase. Theca lutein cells demonstrated a diffuse cytoplasmic staining which was less intense than that observed in granulosa lutein cells. Staining appeared less intense in tissues from the early or late phase. Ovarian stromal cells demonstrated little or no PGHS immunoreactivity. PGHS staining in the corpus luteum of pregnancy was similar in intensity and cell distribution to that of mid-luteal corpus luteum. In summary, human corpus luteum contains immunoreactive PGHS which localized mainly to well-differentiated granulosa lutein cells.     

The corpus luteum of the rhesus monkey (Macaca mulatta) during Late pregnancy. An electron microscopic study

Fine structural observations on the corpus luteum of late pregnancy in rhesus monkeys (Macaca mulatta) are described. Ovariectomy was performed between 141 and 146 days of pregnancy. Granulosa lutein cells measured 25 to 30 μ and were supplied by fenestrated capillaries. The plasma membranes of neighboring cells formed intercellular canaliculi which were bordered by microvilli. The nuclei were large and contained a nucleolus and peripheral heterochromatin. The central region of the cytoplasm contained a high concentration of: (1) rod-shaped mitochondria with tubular cristae and some electron-opaque inclusions in their matrices, (2) elaborate Golgi complexes and associated large tubular cisternae, and (3) tubular as well as cisternal agranular endoplasmic reticulum. The more peripheral region of the cytoplasm was filled with: (1) well developed parallel arrays of granular endoplasmic reticulum, (2) bundles of 50 Å filaments with dense areas, and (3) dispersed lipid droplets. Membrane-bound granules of various types were observed in most cells. Large lacunae were present in the cytoplasm which were not confluent with the intercellular canaliculi or the perivascular spaces. Microvilli projected into the empty-appearing lumen of the lacunae. It was concluded that the morphological features of the granulosa lutein cells of late pregnancy were consistent with those considered to be necessary for steroidogenic function.