Search

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3β-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3β-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23μm) and LLC (range 25-55μm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3β-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Immunoexpression of androgen receptor (AR) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was investigated in three generations of corpora lutea (CLs), found in the ovaries of rats on Days 1, 2, 5, 9, 14 and 20 of pregnancy. The youngest generation of CLs functioned during the whole pregnancy, whereas the older and the oldest generations underwent earlier regression. The newly formed CLs exhibited weak cytoplasmic 3beta-HSD expression. During subsequent days, a gradual increase in 3beta-HSD immunolabelling was observed, followed by a decrease on Day 20. In the older and the oldest CLs, surviving luteal cells demonstrated strong, although in the oldest CLs mostly perinuclear, 3beta-HSD immunoreaction. The newly formed CLs showed weak nuclear AR immunolabelling, which became stronger during the following days. On Day 20, luteal cells demonstrated a weaker nuclear immunoreaction. The older and oldest generations of CLs exhibited weaker and almost negative AR immunolabelling, respectively. Of special interest was the richly vascularised apical region of young CLs. Here luteal cells with more intensive 3beta-HSD staining predominated, whereas cytoplasmic AR immunoreaction was accompanied by positive or negative nuclear AR immunoexpression. The present studies showed differences in AR and 3beta-HSD distribution within various generations of CLs and within particular regions of the same young CL.

The Japanese black bear, Ursus thibetanus japonicus, is a seasonal breeder and shows delayed implantation for several months during pregnancy. The objective of this study was to clarify the steroidogenic capability of the corpus luteum and placenta during pregnancy, including both delayed implantation and fetal development, by immunolocalization of steroidogenic enzymes in these organs of the Japanese black bear. Ovaries and placentae from 15 wild Japanese black bears, which had been killed legally by hunters and were thought to be pregnant, were used in an immunocytochemical study to localize the cholesterol side chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) by the avidin-biotin-peroxidase complex method using polyclonal antisera raised in mammals against P450scc, 3betaHSD, P450c17 and P450arom. P450scc and 3betaHSD were localized in all luteal cells throughout pregnancy. P450c17 was present in a few luteal cells, especially in the outer area of the corpus luteum throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. Cells positively immunostained for P450c17 were significantly smaller than negatively immunostained cells (P < 0.01). P450arom was present sporadically in a few luteal cells throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. The size of cells positively immunostained for P450arom was not significantly different from that of negatively immunostained cells. The whole placenta was negatively immunostained for P450scc, 3betaHSD and P450c17, but P450arom was present in the syncytiotrophoblasts and endothelial cells of maternal blood vessels. These results indicate that, in the Japanese black bear, corpora lutea are a source of progesterone which may play an important role in the maintenance of delayed implantation and fetal development during pregnancy. Corpora lutea have a minimum capability to synthesize androgen in small luteal cells and oestrogen in normal-sized luteal cells during pregnancy, and placentae have the ability to synthesize oestrogen during late pregnancy.

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

We tested the hypotheses that bovine high-density (HDL) and low-density (LDL) lipoproteins differentially influence cellular proliferation and progesterone and IGF-I production by bovine small and large luteal cells. Unit gravity sedimentation was used to produce enriched cultures of small (> 95% pure) and large (75 to 90% pure) luteal cells from corpora lutea (CL) on d 4 and 10 of the estrous cycle. Addition of LDL, HDL, or both resulted in the maintenance of higher (P < .05) numbers of 3beta-hydroxysteroid dehydrogenase (HSD)-positive small and large cells in culture and produced a marked proliferation of 3beta-HSD-negative small luteal cells compared to control medium. Low-density lipoprotein and HDL each stimulated greater (P < .01) progesterone secretion in enriched large cell cultures on both days of the cycle, and by small luteal cells on d 10, compared to the control. Together, LDL and HDL maximized this response. Lipoproteins markedly stimulated (P < .01) the secretion of IGF-I by bovine large luteal cells, and secretion was greater (P < .05) by cells from d 10 CL compared to d 4 CL. Results suggest that the actions of lipoproteins in bovine luteal cells extend beyond their widely recognized roles in steroidogenesis and include remarkable effects on cellular proliferation and IGF-I secretion. Type of lipoprotein (LDL vs HDL) did not have differential effects on any of the variables measured.

The onset of equine chorionic gonadotrophin (eCG) secretion in pregnant mares is associated with an increase in luteal androgen and oestrogen production. The luteal cell type(s) responsible for the increased production of androgens and oestrogens has not been identified in the equine corpus luteum. In this study, we examined the pattern of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450(17 alpha)) and cytochrome P450 aromatase (P450arom) by immunohistochemistry in equine luteal tissue collected during dioestrus (days 7-10; n = 4) and early pregnancy, before (days 29-35; n = 4) and after (days 39-45; n = 4) the onset of endogenous eCG secretion. All luteal cells expressed 3 beta-HSD, P450(17 alpha) and P450arom. The distribution of 3 beta-HSD, P450(17 alpha) and P450arom did not differ with stage of the reproductive cycle. The intensity of immunohistochemical staining for 3 beta-HSD did not appear to differ with reproductive stage. In contrast, the intensity of immunostaining for P450(17 alpha) increased after the onset of eCG secretion. The intensity of immunostaining for P450arom increased during pregnancy before the onset of eCG secretion and diminished after the onset of eCG secretion to the intensity seen in dioestrous corpora lutea. This finding suggests that androgen and oestrogen production is not compartmentalized within the equine corpus luteum. Both large and small luteal cells express the steroidogenic enzymes necessary for oestrogen production, and the intensity of immunostaining for P450(17 alpha) and P450arom appears to be stage-specific.

Twenty nonlactating beef cows were used to determine the effects of dietary energy restriction on ovarian follicular and corpus luteum (CL) development. Cows were fed to either gain (controls) or lose (restricted; RES) body weight. Observations continued until RES cows developed a subfunctional CL (progesterone [P4] < 1.5 ng/mL on d 10 of a cycle; n = 4) or had functional CL (P4 > or = 1.5 ng/mL on d 10 of a cycle; n = 6) followed by anestrus, at which time observations were discontinued on individual controls. Estrous cycles were then standardized for all cows. For RES cows developing subfunctional CL, cycle A was the cycle before development of the subfunctional CL, and cycle B was the 11-d period during development of a subfunctional CL. For RES cows with a functional CL, cycle A was the next to last cycle before anestrus, and cycle B was the 11-d period during formation of a functional CL. Daily P4 concentrations did not differ (P > .10) between controls or RES cows developing functional CL during cycle B but were lower (P < .05) in RES cows developing subfunctional CL. Ovulatory follicles and CL were smaller (P < .05) in RES cows during cycles A and B compared with controls. Daily IGF-l concentrations were higher on d 2 through 4 of both cycles in RES cows developing functional CL compared with RES cows developing subfunctional CL (P < .05). Feeding diets limited in energy resulted in two types of CL. These differences may have been due to IGF-I concentrations.

Endothelial cells have common as well as specialized roles in different tissues and organs; as such, the abundant endothelial cells in the corpus luteum (> 50% of total cell population) could have unique activities necessary for luteal function. Our objective was to establish a method for isolating a pure population of endothelial cells from the primate corpus luteum and to determine the basic conditions for in vitro culture. Corpora lutea collected from rhesus monkeys throughout the luteal phase of the menstrual cycle were minced and enzymatically dispersed into single cell suspensions. Endothelial cells were isolated from the remaining cells (i.e., steroidogenic, fibroblastic, etc.) utilizing magnetic beads labeled with a lectin, Ulex europaeus agglutinin-1 (UEA-1), which binds a sugar found only on primate endothelial cells. After exposure to a magnetic field, UEA-1-negative (-) cells were decanted from the pelleted UEA-1-positive (+) cells; to remove beads from the UEA-1 (+) cells, excess sugar was applied. After optimization of the bead-to-cell ratio, the UEA-1 (+) group contained a population of cells 8-12 microns in diameter (typical endothelial cell size) and < 1% steroidogenic cells. UEA-1 (-) cells were larger (15-35 microns) and stained histochemically for the steroidogenic marker, 3 beta-hydroxysteroid dehydrogenase. Immunocytochemical analysis demonstrated that > 93% of all cells in the UEA-1 (+) group stained positive for the specific endothelial cell marker, platelet/endothelial cell adhesion molecule-1. Cultured UEA-1 (+) cells produced low levels of progesterone and were unresponsive to hCG (100 ng/ml). In contrast, cultured UEA-1 (-) and mixed (unsorted) cells produced high basal levels of progesterone and exhibited a > 3-fold increase in response to hCG treatment. Preliminary experiments comparing different culture media and matrices demonstrated that 1) cell proliferation was unaffected by type of medium (i.e., Dulbecco's Modified Eagle Medium [DMEM]/ F12 or McCoy's 5A); 2) the presence of serum was essential in the absence of added growth factors, and 3) extracellular matrix had a profound effect on proliferation. UEA-1 (+) cells exhibited a dose-dependent increase in cell proliferation in response to vascular endothelial growth factor (VEGF) in the presence and absence of fetal calf serum. In the absence of serum, VEGF stimulated proliferation of UEA-1 (+) cells plated on fibronectin but not collagen I, whereas in the presence of 10% fetal calf serum, both matrices supported VEGF-induced mitogenesis. These studies provide for the first time an efficient, reliable method for isolating primate luteal endothelial cells and describe in vitro culture conditions for subsequent studies examining luteal endothelial cell function and regulation.

The effects of somatotropin (ST) on functions of porcine corpora lutea (CL) during pregnancy were investigated. Twenty-four crossbred (Yorkshire/Landrace) gilts from d 30 to 43 of pregnancy were injected daily with 5 mg of recombinant porcine somatotropin (rpST; n = 12) or 1 mL of saline (control, n = 12). Blood was collected on d 30, 37, and 43 for analyses of plasma progesterone. Gilts were killed on d 44 of pregnancy, and mRNA were isolated from CL, ovary, and liver. Messenger RNA expression for LH receptor, FSH receptor, ST receptor, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and cytochrome P450 side-chain cleavage enzyme (SCC) were measured. Liver, CL, and ovary contained a 4.7-kb mRNA of ST receptor, but the liver contained more mRNA for ST receptor than did CL or ovary (.97 +/- .18, .47 +/- .04, and .25 +/- .04 units, respectively). There were two variants of LH receptor mRNA in CL (6.8 and 4.4 kb). The CL also contained a 1.8-kb mRNA of SCC and a 1.7-kb mRNA of 3 beta-HSD. No FSH receptor mRNA was detected in CL of the pig. The rpST treatment did not affect the mRNA level of ST receptor, 3 beta-HSD, SCC, or 4.4-kb mRNA of the LH receptor. The 6.8-kb mRNA for the LH receptor was decreased (P < .05) by rpST (.56 +/- .04 vs .78 +/- .05 units). Furthermore, concentrations of plasma progesterone decreased (P < .001) in gilts treated with rpST. Decreased luteal function was associated with decreased expression of LH receptor in rpST-treated gilts. The luteotropic effects of ST observed in vitro do not necessarily occur in vivo when gilts are administered rpST during pregnancy.

In this study the luteotropic activity of mouse placental lactogen I (mPL-I) at midpregnancy was assessed using in vivo and in vitro methodologies. Ovaries from 10-day pregnant mice were enzymatically dispersed and plated on fibronectin-coated wells in serum-free medium. The percentage of ovarian cells that stained for the presence of 3 beta-hydroxysteroid dehydrogenase activity was 24.4 +/- 2.7% at the time of plating and remained constant (26.1 +/- 5.0%) after a 20-h attachment period. Two types of 3 beta-hydroxysteroid dehydrogenase-staining cells, with distinct differences in size and morphology, were present in the culture. Large luteal cells (26-45 microns) were characterized by a small round nucleus and spherical shape with abundant cytoplasm. In contrast, small luteal cells (< 20 microns) were stellate, with little cytoplasm and a large oval nucleus. Basal progesterone secretion was maintained without a change in cellular DNA content and cell number for 168 h of culture. Treatment of ovarian cells with mPL-I (0.05-10 micrograms/ml) caused a dose-dependent increase in the progesterone concentration in the medium. The magnitude and time course of mPL-I-stimulated progesterone accumulation in culture were dependent on the time after plating that mPL-I treatment was initiated. The effects of mPL-II and mouse PRL (mPRL) on progesterone production were similar to those of mPL-I. The ability of sera from 10-, 14-, and 17-day pregnant mice to maintain progesterone production in bromocryptine-treated hysterectomized mice was also examined. Mice were hysterectomized on day 9 of pregnancy, and serum progesterone, mPL-I, mPL-II, and mPRL concentrations were measured 72 h later. Twice daily injections of 0.5 ml day 10 pregnancy serum maintained the circulating progesterone concentration at values not different from those present at the time of hysterectomy. In contrast, serum progesterone concentrations were not maintained in mice treated with serum of 14- or 17-day pregnant mice or with saline. Depletion of mPL-I from day 10 pregnancy serum by affinity chromatography on an anti-mPL-I column removed all luteotropic activity, as determined by the inability of this modified serum to maintain the serum progesterone concentration in bromocryptine-treated hysterectomized mice. A similar pool of day 10 pregnancy serum chromatographed on a nonspecific IgG control column did maintain progesterone production, but at somewhat lower concentrations than those present at the time of surgery. These studies offer direct evidence that mPL-I and mPL-II are luteotropic and support progesterone production at midpregnancy in the mouse.

Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.

Corpora lutea were obtained from mares at days 3, 10 and 14 after ovulation, and examined histologically. The morphology of isolated luteal cells obtained by either mechanical or collagenase dissociation of the tissue was examined and the cells stained to detect the steroidogenic enzyme delta 5, beta-hydroxysteroid dehydrogenase. The ratio of large:small cells was significantly higher for cells obtained from mechanically dissociated luteal tissue than for cells obtained by collagenase dissociation (P < 0.01). Cells obtained by both mechanical and collagenase dissociation secreted progesterone, although neither cell population responded to exogenous gonadotrophin with an increase in progesterone secretion. Homogenates of equine luteal tissue bound 125I-labelled human LH with high affinity and specificity, and the specific activity and binding affinity of luteal LH receptors did not change significantly from day 3, to days 10 and 14 after ovulation. However, mechanically dissociated cells on days 10 and 14 bound significantly more LH than did collagenase-dissociated cells on these days (P < 0.05). These results indicate that (i) collagenase dissociation of mare luteal tissue yields a population of cells that is unrepresentative of the corpus luteum, and (ii) the mare corpus luteum is not responsive to LH in vitro at the stages examined.

The objective was the immunocytochemical localization of steroidogenic enzymes in the corpus luteum of Hokkaido brown bears during the period of delayed implantation. Cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) were localized as biosynthetic sites of pregnenolone, progesterone, androgens, and oestrogens, respectively. Ovaries containing corpora lutea were obtained from three mature bears during the expected delayed implantation period and ovarian sections were immunostained by the avidin-biotin-peroxidase complex method using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. P450scc and 3 beta HSD were localized in all luteal cells, whereas P450c17 (0.4-5.1% of 1000 cells) and P450arom (7.1-11.2% of 1000 cells) were localized in only a few luteal cells. These data suggest that luteal cells contain steroidogenic enzymes required for progesterone synthesis but also have a minimum capability for synthesizing androgen and oestrogen during the delayed implantation period in Hokkaido brown bears.

Human luteal cells have characteristic organelles, such as well developed smooth endoplasmic reticulum (sER), large mitochondria with tubular cristae, lysosome like granules and lipid droplets. To investigate possible roles of these characteristic organelles, 21 human active corpora lutea, obtained from women aged 32-46, were studied cytochemically on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome c oxidase (CY-O), NADPH diaphorase (NP-DP) and acid phosphatase (ACP) activities, using the methods by Benkoël et al., Ogawa et al., Ago et Ogawa and Barka et Anderson. Subcellular localization of 3 beta-sterol were also examined using the 3 beta-sterol digitonin complex method by Mizuhira et al., 3 beta-HSD and NP-DP activities were demonstrated in the intercrystal and outer space of mitochondria and sER. CY-O activities were demonstrated in mitochondria. ACP activities were demonstrated in lysosomes and Golgi apparatus. Some lipid droplets contained the crystals of 3 beta-sterol digitonin complex. These data suggest that human luteal mitochondria can synthetize progesterone from cholesterol, and that mitochondrial respiratory chain activities in human luteal cells is highly elevated. Human luteal cells probably require much oxygen. Elevated ACP activities indicate that lysosomes play a role in the steroidogenesis. Some lipid droplets may contain cholesterol and other 3 beta-sterols.

In corpora lutea of pregnancy of dairy cows delta 5-3 beta-hydroxysteroid dehydrogenase and succinate dehydrogenase were demonstrated histochemically and evaluated densitometrically. Serum progesterone was determined radioimmunologically. Activities per volume unit of delta 5-3 beta-hydroxysteroid dehydrogenase and succinate dehydrogenase in large and small luteal cells as well as progesterone concentrations, exhibited no typical and correlated pattern during pregnancy. Large luteal cells in regressive tissue regions showed weaker delta 5-3 beta-hydroxysteroid dehydrogenase activities than in maturing or well-developed tissue regions. Succinate dehydrogenase activities of small luteal cells were highest in regressive luteal tissue. The results indicate that structural development of bovine luteal tissue during pregnancy is reflected by corresponding enzyme activities.

This study was designed to test the hypothesis that the corpus luteum of primate species consists of cell subpopulations that differ in physical characteristics, function, and regulation by endocrine and paracrine factors. The corpus luteum (n = 25) was removed from rhesus monkeys at the mid-luteal phase of the menstrual cycle (Days 7-8 after the surge of luteinizing hormone, LH) and enzymatically dispersed. Freshly dispersed cells were analyzed and sorted on the basis of their forward and 90 degrees light scatter (FLS and 90LS, respectively) properties using an EPICS C flow cytometer. Freshly dispersed and sorted cells were fixed, stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and measured to determine their diameters. Freshly dispersed (MIX) and sorted cells from corpora lutea during the early (Days 4-5 after the LH surge; n = 4) and mid-luteal phases of the cycle were incubated in vitro and steroid production was assessed. The size distribution of dispersed cells revealed four peaks that corresponded to small (10-15 microns in diameter) 3 beta-HSD-negative, and small, medium (16-20 microns), and large (greater than 20 microns) 3 beta-HSD-positive cells. Analysis of dispersed cells for FLS and 90LS demonstrated two continua (C alpha and C beta). C alpha contained single cells and cell clusters; 99.7 +/- 0.3% (n = 3) of the cells were less than or equal to 15 microns in diameter and 96.7 +/- 0.3% were 3 beta-HSD-negative. C alpha cells produced low levels of progesterone (0.2 +/- 0.1 ng/ml per 5 x 10(4) cells; n = 3) in vitro under basal conditions. C beta consisted of single cells from 10 microns to 40 microns in diameter and contained the lipid-filled and 3 beta-HSD-positive cells. Two regions (R1 and R3) of C beta were defined and their cells separated. In R1, 96 +/- 2% (n = 3) of the cells had diameters of less than or equal to 15 microns, whereas 82 +/- 4% (n = 3) of those in R3 were greater than or equal to 20 microns. Basal progesterone production by R3 cells from early luteal phase of the cycle was 12 times greater than that by R1 cells (n = 3 per group).(ABSTRACT TRUNCATED AT 400 WORDS)

3 beta-hydroxysteroid dehydrogenase (3 beta-HDS) activity was demonstrated ultrastructurally in human corpora lutea obtained from the women aged 32-46 who were treated by the hysterectomy for various gynecological indications. The corpora lutea were removed from ovaries and cut into small pieces of 1 mm3. Specimens were incubated at 25 degrees C for 20 min. in the media containing dehydroepiandrosterone 0.6 mg, 0.1 M sodium citrate 0.3 ml, 5 mM potassium ferricyanide 1 ml, 30 mM copper sulfate 1 ml, N.A.D. 3.6 mg, sucrose 1.0 g and 0.1 M phosphate buffer (pH 7.2) 6 ml. For a control study, 1.5 mg cyanoketone (by the courtesy of Dr G.O. Pott), the specific inhibitor for 3 beta-HSD, was added to the media. The materials were fixed in 4% glutaraldhyde and 1% OsO4 and embedded in Epon 812. Electron dense reaction products of copper ferrocyanide were observed in smooth endoplasmic reticulum (sER) and in intercristal and outer space of mitochondria. In control studies, 3 beta-HSD activity were completely abolished. It is concluded that not only sER but also mitochondria has 3 beta-HSD activity in human corpora lutea. Furthermore, mitochondria seems to have capability to synthetize progesterone from cholesterol by itself.

The capacity for steroid production in the corpora lutea (CL) of bank voles was examined by enzyme histochemistry for 3 beta-hydroxysteroid dehydrogenase. The results revealed similar activity in the CL resulting from sterile matings and the CL of pregnancy 2 days after mating. Six days after mating, the CL of pregnancy showed a strong reaction, while there was almost no reaction in the CL from sterile matings, which were clearly regressed at this time. It is suggested that the CL from sterile matings produce progesterone before regression and that this progesterone may prime the luteotrophic complex, increasing fertility of subsequent matings.