Programmed cell death in human ovary is a function of follicle and corpus luteum status

Although extensive investigation on follicular apoptosis (programmed cell death) has been conducted in the infraprimate ovary, there is little information regarding apoptosis and its relationship to follicular status in the human. In this study, apoptosis was investigated in 116 human ovarian follicles (primordial to dominant) and 5 corpora lutea from a total of 27 premenopausal women. Follicles and corpora lutea were evaluated for the presence of DNA fragmentation, characteristic of apoptosis, by two methods: in situ hybridization using 3' end-labeling of DNA with digoxigenin-labeled nucleotides and subsequent digoxigenin antibody and peroxidase staining, and/or biochemical analysis of low molecular weight DNA laddering. Follicle functional status was evaluated by determining follicle sizes and follicular fluid androgen/estrogen (A/E) ratios. No apoptosis was observed in 67 primordial, primary, or secondary follicles. Positive staining for DNA fragmentation was found in a few granulosa cells in 0.1- to 2-mm follicles, whereas abundant staining in granulosa was detected in 2.1- to 9.9-mm follicles. In contrast, no DNA fragmentation was detected in dominant follicles (10-16 mm). The frequency of apoptosis in follicles was calculated to be 37% in 0.1- to 2-mm follicles, 50% in 2.1- to 5-mm follicles, and 27% in 5.1- to 9.9-mm follicles. Abundant low molecular weight DNA laddering was only found in androgen-dominant follicles and not in estrogen-dominant follicles. Positive staining for DNA fragmentation and low molecular weight DNA laddering were observed in degenerating but not healthy-appearing corpora lutea. In the former, DNA fragmentation was found primarily in large luteal cells. These data suggest that follicular atresia in human ovary results from normal programmed cell death and primarily occurs in the granulosa cell layers of the early antral and < 10-mm antral follicles primarily. Furthermore, because apoptosis occurs as early as the 200-mm stage, follicle selection may begin as early as the initial formation of the antrum. The results also suggest that degeneration of the corpus luteum occurs by apoptotic mechanisms.     

Endothelial cell proliferation follows the mid-cycle luteinizing hormone surge, but not human chorionic gonadotrophin rescue, in the human corpus luteum

The corpus luteum is essential for the maintenance of early pregnancy in women. Angiogenesis may be one factor involved in luteal rescue. The aim of this study was to determine the changes in endothelial cell proliferation throughout the luteal phase and in human chorionic gonadotrophin (HCG)-simulated early pregnancy. Human corpora lutea obtained throughout the luteal phase and in simulated early pregnancy were immunostained with antibodies for endothelial and proliferating cells. Number and distribution of endothelial and proliferating cells were examined. Endothelial cells were least abundant in the early luteal phase, increasing in the mid-luteal phase (P < 0.03). Endothelial numbers did not differ significantly between the late and the rescued corpora lutea. Endothelial cell proliferation was greatest in the early luteal phase and continued at a lower level during later stages. Simulated early pregnancy resulted in no change in endothelial cell proliferation. These results showed that a high degree of endothelial cell proliferation is associated with formation of the human corpus luteum. Unchanging levels of proliferation following HCG treatment (for 5-8 days from day 12 to day 16 post-ovulation, at 125 IU to 16,000 IU, following a daily doubling of dose) suggest that alternative processes are involved during luteal rescue.     

Macrophages are the major source of tumor necrosis factor alpha in the porcine corpus luteum

This study was designed to determine the source of tumor necrosis factor (TNF) alpha within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNFalpha, anti-porcine macrophage-specific antigen, or anti-alpha-actin (marker of pericyte and smooth muscle cells). Dolichos biflorus lectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNFalpha was apparent in porcine CL throughout the estrous cycle. TNFalpha immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNFalpha in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNFalpha secretion by each subpopulation was determined by measuring bioactive TNFalpha in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNFalpha (up to 400 pg/10(6) cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/10(6) cells) of bioactive TNFalpha. Large luteal cells secreted slightly greater amounts of TNFalpha (10-15 pg/10(6) cells). Local macrophages appear to be the primary source of TNFalpha in the porcine CL.     

Ultramicroscopic studies on the human corpus luteum menstruationis and graviditatis

In high active steroid producing cells of the human corpus luteum we can find in the cytoplasm a well developed smooth endoplasmic reticulum in form of smooth tubules and homogenously spread vesicles resulting from an active metabolic process. We also find specific concertinalike folded membrane complexes which too belong to the endoplasmic reticulum. Numerous mainly tubular mitochondria contain dense lipoid inclusions. Their close conection with the endoplasmic vesicles is expression of a direct interaction of both cell organelles in progesterone production. The transport of the intracellular formed steroid hormones to the capillary takes place via locally enlarged intercellular spaces which communicate with the pericapillary space. On the other hand there are pericellular canaliculi which are coated with a membrane. They have no direct connection to the pericapillar space.     

Focally regulated endothelial proliferation and cell death in human synovium

Angiogenesis and vascular insufficiency each may support the chronic synovial inflammation of rheumatoid arthritis. We have shown by quantitative immunohistochemistry and terminal uridyl deoxynucleotide nick end labeling that endothelial proliferation and cell death indices were each increased in synovia from patients with rheumatoid arthritis compared with osteoarthritic and noninflamed controls, whereas endothelial fractional areas did not differ significantly among disease groups. Markers of proliferation were associated with foci immunoreactive for vascular endothelial growth factor and integrin alpha(v)beta3, whereas cell death was observed in foci in which immunoreactivities for these factors were weak or absent. No association was found with thrombospondin immunoreactivity. The balance between angiogenesis and vascular regression in rheumatoid synovitis may be determined by the focal expression of angiogenic and endothelial survival factors. Increased endothelial cell turnover may contribute to microvascular dysfunction and thereby facilitate persistent synovitis.     

Differences of microvascular endothelium in the bovine corpus luteum of pregnancy and the corpus luteum of the estrous cycle

A haemolysin produced by Actinomyces pyogenes ATCC 8164 was purified from culture supernatant by ammonium sulphate and polyethylene glycol precipitation, ion-exchange chromatography on DEAE-Sephacel, and fast-protein-liquid-chromatography on Superose 12 prep grade. The purified haemolysin, designated as pyolysin, displayed a single band on poly-acrylamide gel electrophoresis, indicating a molecular weight of 55000. Additionally, using gel filtration, the same molecular weight was estimated. Further studies of the eluate of ion-exchange chromatography using isoelectric focusing also revealed a single protein band at pH 9.38 with haemolytic activity. A specific antiserum produced against pyolysin inhibited the haemolytic activity. The purity of the isolated protein was also determined by Western Blot analysis with antiserum obtained from a cow inoculated with culture supernatant from A. pyogenes and Peptococcus indolicus. The isolated pyolysin appeared to be heat-labile and displayed cytotoxic effects on poly-morphonuclear leucocytes and on pTK2 kidney cells.     

Relaxin: a product of the human corpus luteum of pregnancy

Plasma samples from peripheral and ovarian veins were obtains from women at cesarean section. A peptide that immunologically cross-reacts with a specific antiserum to porcine relaxin is present in all samples. Its concentration is four times higher in the ovarian vein draining the ovary, which contains the corpus luteum of pregnancy, than in either the peripheral vein or the contralateral ovarian vein. Secretion of ovarian relaxin correlates with secretion of ovarian progesterone, thus providing another index of luteal function.     

Ludwig Fraenkel, corpus luteum and discovery of progesterone]

In 1934, after a dramatic neck-and-neck scientific race, four research groups independently from each other reported on the successful purification of progesterone. Two of the groups were from the then-German cities of Breslau and Danzig, the others were from the USA and Switzerland. Possibly, the Breslau group had already had the purified hormone as early as 1933. At that time, gynecologist Ludwig Fraenkel (1870-1951) had been their "spiritus rector" for more than three decades. It was Fraenkel himself who at the beginning of the century, in examining a hypothesis of the anatomist Gustav Jacob Born (1851-1900), had provided experimental proof for an endocrine function of the corpus luteum. Later on, Fraenkel enlisted the help of chemist Karl Heinrich Slotta (1895-1987) in the purification of the hormone. This took place after important requirements for the isolation and for the semiquantitative determination of the hormone (e.g. the Corner-Allen-Test) had been established elsewhere. Also belonging to the Breslau research group were Erich Fels (1897-1981) and Heinrich Ruschig (born in 1906). Fels was an assistant to Fraenkel, Ruschig a PhD-candidate directed by Slotta. Shortly after the group had succeeded in purifying Progesterone the Breslau group was broken apart by the National Socialists' racial policies: Fraenkel, Fels and Slotta were forced into emigration.     

Volume of luteal tissue and concentration of serum progesterone in cows bearing homogeneous corpus luteum or corpus luteum with cavity

Ultrasonographical examinations of ovarian structures were performed in 27 inseminated cows at estrus days and on days 4, 9, 20, 25, 30, and 40 after ovulation. Three cows were used twice. Corpora lutea (CLs) with a cavity were compared with homogeneous CLs. in pregnant and nonpregnant cows. Diameters and volumes of CLs and cavities, as well as volumes of luteal tissue and concentrations of serum progesterone were determined. The volumes of the structures were calculated using a mathematical formula for a rotary ellipsoid. Homogeneous CLs and CLs with a cavity and their luteal tissue reached a maximum volume in nonpregnant and pregnant cows on day 9 after ovulation. At this time, CLs volumes were 7.52 +/- 3.14 (homogeneous CLs, n = 4) and 4.54 cm3 (CLs with a cavity, n = 1) in nonpregnant cows, and 6.05 +/- 1.71 (homogeneous CLs, n = 10) and 9.54 +/- 2.67 cm3 (CLs with a cavity, n = 15) in pregnant cows. The volumes of luteal tissue were 7.52 +/- 3.14 and 4.33 cm in nonpregnant cows and 6.05 +/- 1.71 and 8.62 +/- 3.46 cm3 in pregnant cows. Concentrations of progesterone in peripheral blood in pregnant cows bearing a homogeneous CLs or CLs with a cavity on day 9 were 3.15 +/- 0.69 ng ml-1 and 4.12 +/- 1.28 ng ml-1, respectively. The concentrations of progesterone were higher in pregnant cows in comparison with nonpregnant cows. CLs with a cavity in pregnant cows contained a higher volume of luteal tissue and higher secretory activity compared to homogeneous.     

Specific non-genomic, membrane-localized binding sites for progesterone in the bovine corpus luteum

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.     

Interaction between two luteal cell types from the corpus luteum of the sow in progesterone synthesis in vitro

Populations of two types of luteal cell (large and small) were prepared from CL of sows at 60 days of gestation. When the two types were recombined and incubated for 2 h, the amount of progesterone released into the medium was almost twice the sum of that released by each cell type alone. When the cells were superfused in vitro, in an "in series" arrangement such that the superfusate from one cell type then passed through the chamber containing the second cell type, the small cells were responsible for increasing the production of progesterone by the large cells. When cell population were superfused with media containing inhibitors of progesterone synthesis, trilostane completely blocked progesterone release but aminoglutethimide only reduced progesterone output by half, even when the concentration of inhibitor was increased. Addition of pregnenolone to the superfusion medium increased progesterone production by both cell types approximately 3-fold, whereas addition of cholesterol only increased progesterone production by the large cells.     

Ontogeny of stem cell factor receptor (c-kit) messenger ribonucleic acid in the ovine corpus luteum

Stem cell factor (SCF) is a pleiotropic growth factor that is expressed by the ovine corpus luteum throughout its life span by both small and large steroidogenic cells. Determination of the action of SCF, however, requires localization of its receptor, c-kit; therefore, the objectives of the present study were to identify and localize c-kit within corpora lutea. Two cDNAs encoding different portions of the c-kit molecule were amplified by the polymerase chain reaction. The first was a 558-base pair (bp) cDNA encoding portions of the transmembrane and tyrosine kinase domains; the second was a 632-bp cDNA encoding most of the ligand-binding domain. Expression of c-kit was quantified by RNase protection assay of total cellular RNA collected on Days 3, 7, 10, 13, and 16 (n = 4, 4, 5, 4, and 4 per group, respectively) of the estrous cycle (Day 0 = estrus). The level of c-kit mRNA was low early in the luteal phase, reached (p < 0.05) maximum levels on Day 13, and then decreased (p < 0.01) on Day 16. On Day 3 (n = 4), c-kit was expressed in a cell-specific manner throughout the corpus luteum; identity of the specific cell types expressing c-kit could not be determined at this stage. On Day 14 (n = 4), c-kit did not appear to be expressed within large luteal cells but was prominently expressed in cells that surrounded large luteal cells and that possessed the morphological characteristics of small luteal cells and endothelial cells. Given the temporal regulation of c-kit expression within the corpus luteum, these data suggest that luteal SCF may act locally.     

Macrophages and methylthio groups in lymphocyte proliferation

Macrophages are shown to replace methylthio disulfides in supporting in vitro proliferation of three cell lines previously characterized as methylthio-dependent. Macrophages have the capacity to generate methylthio groups from methylthioadenosine. It is hypothesized that macrophages stimulate cell proliferation both in normal immune systems and in certain cancers by providing an abundance of methylthio groups. Fetal calf serum is shown to contain methylthio groups. It appears that, in cell cultures containing fetal calf serum, sulfhydryl compounds stimulate cell proliferation by making the methylthio groups in the serum available to the cells.     

Effect of gonadotrophin on cell and matrix retention and expression of metalloproteinases and their inhibitor in cultured human granulosa cells modelling corpus luteum function

A retrospective cohort study in 1794 male ceramic workers in the Netherlands was carried out to analyze the lung cancer risk in relation to crystalline silica exposure and silicosis. They had all been employed for two years or longer in ceramic industries between 1972 and 1982. During a health survey, 124 cases of simple pneumoconiosis were diagnosed; after 14 years of follow-up, 161 deaths had occurred. No increased overall and cause-specific mortality was found in the total group of ceramic workers, and a statistically significant cumulative dose-response relation for silica exposure and lung cancer did not emerge. An excess lung cancer mortality appeared among workers with simple pneumoconiosis. The authors conclude that the disease process resulting in silicosis in the ceramic industry carries an increased risk of lung cancer, which is supportive of a nongenotoxic pathway.     

Relationships between estrogen receptor and estradiol-stimulated progesterone synthesis in the rabbit corpus luteum

The effects of estradiol on luteal estrogen receptor and steroidogenesis were examined on Days 9 through 11 of pseudopregnancy. In normal pseudopregnant rabbits, unoccupied cytoplasmic and total nuclear estrogen receptor were 2.6 +/- 0.4 fmol/microgram DNA and 0.4 +/- 0.1 fmol/microgram DNA, respectively, on Day 10 of pseudopregnancy. An i.v. injection of 4 micrograms of estradiol caused the translocation of cytoplasmic receptor and a 6.6-fold increment in total nuclear receptor within 15 min, which was followed by rapid processing of the nuclear receptor. Both cytoplasmic and nuclear estrogen receptor returned to normal values within 24 h, and during this period, serum progesterone did not change significantly. Withdrawal of an estradiol implant from animals on Day 9 of pseudopregnancy initiated a marked decline in serum progesterone within 24 h. Following an injection of saline or of 4 micrograms estradiol on Day 10, unoccupied cytoplasmic estrogen receptor in saline- and estradiol-injected rabbits was 1.0 +/- 0.1 fmol/microgram DNA and 1.9 +/- 0.1 fmol/microgram DNA, respectively, on Day 11 of pseudopregnancy. Associated with the increase in cytoplasmic receptor there was an increase in serum progesterone (8.2 +/- 1.5 ng/ml), in contrast to saline-injected animals in which serum progesterone continued to decline (1.6 +/- 0.4 ng/ml). Despite the significant differences in cytoplasmic receptor and in progesterone synthesis, total nuclear estrogen receptor was not different in these animals. These data suggest that in corpora lutea already secreting progesterone at high rates during midpseudopregnancy, the rapid translocation of available estrogen receptor does not cause further stimulation of progesterone synthesis. However, if steroidogenesis is first reduced experimentally, then an injection of 4 micrograms of estradiol can readily stimulate progesterone synthesis, and this stimulation is associated with an increase in cytoplasmic receptor.