Activity Of Luteal Cells Research Articles

LH Stimulates the Phosphorylation of Key Components of the Wnt Signaling Pathway in Bovine Steroidogenic Luteal Cells.

Luteinizing hormone (LH) stimulates progesterone synthesis in bovine corpus luteum by a cAMP/Protein kinase A (PKA)-dependent mechanism. Previous studies demonstrate that the actions of LH may intersect with glycogen synthase kinase-3B (GSK3B) and β-catenin, key components of the Wnt signaling pathway. In the canonical Wnt signaling pathway, Wnt signaling inactivates GSK3B which prevents the degradation of β-catenin and allows it to translocate to the nucleus to activate target gene expression. Evidence in fibroblast cell lines indicates that PKA can stabilize or activate β-catenin by phosphorylating β-catenin on C-terminus serine residues (S552 and S675). It is not known, however, whether LH regulates the phosphorylation and subcellular localization of β-catenin. In the present study, we tested the hypothesis that LH stimulates the phosphorylation of GSK3B(S9) and β-catenin(S552/S675). Consistent with a role for Wnt signaling components in the action of LH, we hypothesized that the LH-induced increases the phosphorylation of these proteins would be present in perinuclear and nuclear compartments in bovine luteal cells. Bovine corpora lutea of early pregnancy were obtained at slaughter. Isolated steroidogenic luteal cells were prepared and treated with LH or activators of PKA. Western blot revealed that LH (100ng/ml) rapidly stimulated the phosphorylation of GSK3B(S9) and β-catenin(S552/S675); maximal responses were attained within 5 min and phosphorylation responses were maintained for up to 60 min without alterations in the levels of GSK3B or β-catenin. Additionally, LH treatment (1-100 ng/ml for 15 min) stimulated concentration-dependent increases in the phosphorylation of these proteins. The effects of LH were mimicked by 8-Br-cAMP and forskolin, agents that increase PKA activity in luteal cells. Immunofluorescence showed that LH treatment for 15 min increased the phosphorylation of GSK3B(S9) predominantly in the Golgi apparatus and nucleus. Since the Golgi and nuclear fraction are closely associated, an experiment was performed to determine whether the Golgi has role in progesterone production in response to LH. Pretreatment of luteal cells with up to 5 μg/ml brefeldin A (BFA, which interferes with anterograde transport from the endoplasmic reticulum to the Golgi apparatus, and disrupts the Golgi) for 2h had no effect on progesterone production in response to LH treatment (100 ng/ml, 4 h). Immunofluorescence studies also showed that LH treatment for 15 min increased levels of phosphorylated β-catenin (S552 and S675) in the nucleus of luteal cells. Subcellular fractionation and Western blot analysis confirmed findings that the LH-induced increases in phospho-S552 β-catenin were predominately localized to the nucleus of luteal cells. The LH-induced increases in phospho-S675 β-catenin were observed in both cytosolic and nuclear factions of bovine luteal cells. Our findings indicate that in response to LH, key components of the Wnt signaling pathway are phosphorylated and localized to the nucleus. The presence of phosphorylated GSK3B in the Golgi apparatus imply a role in protein trafficking, but our studies with BFA rule out a role in the acute steroidogenic response to LH. The nuclear localization of phosphorylated β-catenin in response to LH may contribute to PKA- and β-catenin-regulated gene expression in steroidogenic luteal cells. Supported by the VA, USDA and the Olson Center for Women's Health.

Immunohistochemical Characterization of S100A6 in the Murine Ovary.

S100 proteins comprise a large family of Ca2+-binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions.

Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells

The cytochrome P450 aromatase (Cyp19) gene encodes an enzyme of crucial importance in the synthesis of estradiol. Estradiol is luteotropic in the rat. In this species, luteal Cyp19 expression increases progressively during pregnancy and falls before parturition. The mechanisms that control these changes are unknown. Using gel shift assays, we sought to identify the promoter regions that control Cyp19 expression in the rat corpus luteum (CL). The Cyp19 promoter contains a cAMP response element-like sequence (CLS), two nuclear receptor elements half sites (NREs), a GATA binding site, a Yin Yang-1 (YY1) response element, and an activation protein 3 (AP3) binding site. Nuclear extracts were obtained from CL of rats on days 4, 15, and 23 of pregnancy and from the ovaries of immature rats treated with vehicle or a hormone that induces Cyp19 expression in the follicles. CLS was active in immature ovaries but inactive in the CL of pregnant rats, whereas binding to NREs and GATA was observed in both tissues. YY1 was inactive in all samples tested. In the CL, AP3 binding was higher on day 15 of pregnancy when compared with day 4 and day 23 but it was absent in ovaries of immature rats, whereas luteinization increased AP3 binding activity. Mutation of the AP3 site blunted the stimulation of Cyp19 promoter activity in granulosa cells. Our results indicate that CLS is active only in follicles; whereas in the CL, binding to the GATA, NRE, and AP3 sites associates with changes in Cyp19 expression, suggesting that they control Cyp19 promoter activity in luteal cells.

Changes in the Distribution of Tenascin and Fibronectin in the Mouse Ovary During Folliculogenesis, Atresia, Corpus Luteum Formation and Luteolysis

Tenascin and fibronectin are components of the extracellular matrices that oppose and promote adhesion, respectively. Using immunohistochemical techniques, we studied the distribution of tenascin and fibronectin in the mouse ovary, in which dynamic reconstruction and degeneration occur during folliculogenesis, atresia, ovulation, corpus luteum formation and luteolysis. In growing follicles, tenascin was only detected in the theca externa layer, while fibronectin was detected in the theca externa layer, theca interna layer and basement membrane. During follicular atresia, granulosa cells, which are surrounded by the basement membrane, began to die through apoptosis. In atretic follicles, tenascin was detected in the basement membrane and theca externa layer. Distribution of fibronectin in atretic follicles was similar to that in healthy growing follicles, except that granulosa cells were slightly immunopositive for fibronectin. In young corpus luteum, luteal cells exhibit high 3 beta -hydroxysteroid dehydrogenase (3 beta -HSD) activity, an enzyme indispensable for progesterone production. Tenascin was barely detected in young luteal cells. 3 beta -HSD activity in luteal cells declines with corpus luteum age, and in older corpus luteum there is an increase in apoptotic death of luteal cells. Tenascin was intensely immunopositive in old luteal cells.In contrast, fibronectin immunostaining in luteal cells was relatively constant during corpus luteum formation and luteolysis. Our observations suggest that tenascin is critical in controlling the degenerative changes of tissues in mouse ovaries. Moreover, in all circumstances observed in this study, tenascin always co-localized with fibronectin, suggesting fibronectin is indispensable for the function of tenascin.

Size distribution of steroidogenic and non-steroidogenic ovine luteal cells throughout pregnancy

AbstractThe present study examines the size distribution of ovine steroidogenic and non-steroidogenic luteal cells throughout pregnancy. Cells were isolated from corpora lutea collected from early (< 8 weeks), mid (9 to 14 weeks) or late (15 to 18 weeks) stages of pregnancy. Cells were stained for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a marker for steroidogenic cells. Both 3β-HSD positive and β-HSD negative cells covered a wide spectrum of size ranging from 7 to 37 μm in diameter. There was a significant increase (P > 0·01) in mean diameter of non-steroidogenic luteal cells as pregnancy progressed. Mean diameter of 3β-HSD negative cells increased from 17·8 (s.e. 0·4) μm in the corpus luteum of early stage of pregnancy to 22·4 (s.e. 0·3) μm in the corpus luteum of advanced pregnancy. However, there was no significant increase in the mean diameter of 3β-HSD positive cells. Corpora lutea obtained from early stages of the pregnancy contained more steroidogenic cells than the cells obtained from mid and late pregnancy (P < 0·01). Percentage of 3β-HSD negative cells had increased 2·07-fold by 18 weeks of pregnancy when compared with the early stage of pregnancy. In contrast, percentage of 3β-HSD positive cells had decreased to 50% of starting values during the same period (P < 0·05). These results indicate that the ovine corpus luteum of pregnancy is morphologically dynamic over the course of pregnancy. Steroidogenic activity of luteal cells may decrease as pregnancy progresses, especially activity of the large luteal cells.

Molecular identification of adenylyl cyclase 3 in bovine corpus luteum and its regulation by prostaglandin F2alpha-induced signaling pathways.

The involvement of cAMP in various aspects of ovarian steroidogenic cells functions has been extensively studied. However, the adenylyl cyclase (AC) types expressed in ovarian cells, of any species, are not yet determined. The present study was undertaken to identify AC types present in bovine luteal cells and their regulation by various stimuli. AC isoforms 2, 3, 5, 6, 7, 8, and 9 were detected in the bovine brain by Northern blotting analysis, whereas the bovine corpus luteum (CL) only expressed AC3 and 6 mRNAs, with AC3 being more abundant than AC6. The use of AC3-specific primers in RT-PCR reaction verified the presence of AC3 mRNA in both bovine and rat CL tissue as well as in bovine steroidogenic luteal cells. Because these two AC isoforms, AC3 and 6, exhibit distinct regulatory patterns we have next examined the effects of various signaling pathways on AC activity in luteal cells. These studies have shown that: 1) prostaglandin (PG) F2alpha and phorbol 12-myristate 13-acetate markedly elevated agonist-stimulated cAMP synthesis (these effects were inhibited by addition of highly specific PKC inhibitor, bisindolylmaleimide); 2) depletion of Ca2+ from the incubation medium inhibited AC activity; 3) physiological concentrations of Ca2+ ions (up to 5 mM) significantly stimulated cAMP production in luteal cells; and 4) the effects of Ca2+ on cAMP synthesis were evident only in the presence of forskolin. These regulatory characteristics of AC activity are consistent with the molecular identification of ACs indicating the presence of AC3 in luteal cells. The reported data may delineate the cross-talk between physiological activators of AC in the CL (such as LH, PGE2, and PGI2) and other ligands (such as PGF2alpha and endothelin-1), which indirectly modulate AC activity. Therefore, the identification of AC isoforms present in luteal cells is an important step toward understanding the mode of action of a wide array of hormones regulating ovarian cells.

Functional lysophosphatidic acid receptor in bovine luteal cells

The aim of this study was to determine whether luteal cells possess functional receptors for lysophosphatidic acid (LPA). We present evidence that [ 3H]LPA binds to a 38–40 kDa protein in a membrane fraction prepared from luteal cells and that this phospholipid is able to induce tyrosine phosphorylation of several proteins (65–125 kDa). Furthermore, LPA upregulates forskolin- and LH/GTP-stimulated adenylyl cyclase activity by changing its Vmax. Although a pertussis toxin-sensitive G-protein has been reported to transmit the inhibitory signals between the LPA receptor and adenylyl cyclase, the observed upregulation of the enzyme activity in luteal cells is not abolished after pre-treating the cells with the toxin, suggesting that a different mechanism is operative in these cells. According to the pharmacological regulatory pattern it is suggested that the modulated adenylyl cyclase isoform is the enzyme subtype V expressed in luteal cells.

Effect of calyculin-A, phosphatase inhibitor, on 20 alpha-hydroxysteroid dehydrogenase activity in rat luteal cell culture.

The effects of calyculin-A (CL-A), a phosphatase inhibitor, on 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in rat luteal cell cultures to examine whether protein phosphatases were involved in the regulation of the enzyme activity. Luteal cells were harvested from the rats on day 6 of pseudopregnancy. In the absence of prolactin (PRL), the stimulatory effect of CL-A (10(-9) M) on 20 alpha-HSD activity was observed during the first 3 h period of the culture, but was not discernible thereafter. Since the enzyme activity of luteal cells during pseudopregnancy is known to be suppressed by PRL, the results suggest that this effect of CL-A could be manifested only as long as the effect of endogenous PRL was remained. On the other hand, CL-A enhanced the 20 alpha-HSD activity of the cells cultured for 24 h, where a spontaneous increase in 20 alpha-HSD activity was blocked by exogenously supplementing the culture medium with PRL. These results suggest that a CL-A sensitive phosphatase(s) may be involved in the action of PRL in suppressing the increase in 20 alpha-HSD activity in rat luteal cells.

Effect of pregnancy, injection of oestradiol benzoate or hCG on steroid concentration and release by pig luteal cells.

Corpora lutea were obtained from pig ovaries on Day 18 of pregnancy or pseudopregnancy. Pseudopregnancy was induced by the administration of oestradiol benzoate on Days 11-15 of the oestrous cycle or by the administration of hCG on Day 12. The luteal cells were prepared for morphometric analysis and investigation of steroid production in vitro by dispersion with 0.25% trypsin. A blood sample from each sow was collected at slaughter for measurement of progesterone, oestradiol-17 beta and testosterone. The concentrations of these steroids were also estimated in luteal tissue and in the medium after incubation. Progesterone concentration was significantly higher (P less than 0.01) in luteal tissue and in plasma of pregnant than of pseudopregnant sows. Testosterone content of luteal tissue from all sows was 20-fold higher than oestradiol, although plasma concentrations of these hormones were not different. The luteal cells from hCG-treated sows produced more progesterone (P less than 0.01) in vitro than did those from the other groups. The luteal cells from oestradiol-treated sows generally released smaller amounts of steroids during incubation. Treatment with hCG increased the proportion of large luteal cells and decreased the proportion of small luteal cells. These results demonstrate that hCG or oestradiol benzoate injections altered the steroidogenic activity of luteal cells and that treatment with hCG was also associated with changes in the diameter of the luteal cells and thus in the ratio of small to large luteal cells.

The effects of peritoneal macrophages on monolayered luteal cell progestin secretion in the rat.

Functionally active or regressing luteal cells were obtained from pseudo-pregnant (psp) rats between days 5-8 of psp or on day 15 of psp, respectively. They were monolayer-cultured (10(6)/dish) in the presence of 0.2 micrograms/ml LH 2.0 micrograms/ml PRL and 10 micrograms/ml pregnenolone for 4 days with or without macrophages, although functionally active luteal cells secreted progesterone dominantly during day 1 of culture (Day 1), the amounts of progesterone and 20 alpha-OH-P secreted were inverted on Day 2, and the dominance of 20 alpha-OH-P continued from Day 2 to Day 4. In the functionally regressing luteal cell culture, more 20 alpha-OH-P than progesterone was secreted throughout the culture period. The addition of peritoneal macrophages (2.5 X 10(6] to the active luteal cell monolayer lengthened the dominance of progesterone secretion for an additional day and the inversion occurred on Day 3. The progestin ratio (progesterone/20 alpha-OH-P) on Day 2 was maintained significantly higher. The daily addition of macrophages maintained the progesterone dominance throughout the culture period. On the other hand, macrophages had no effect on luteal cells already functionally regressing. These results indicate that macrophages are effective in maintaining the progesterone secreting activity of luteal cells in vitro.

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.

Effects of estradiol on calcium-specific protein phosphorylation in the rat corpus luteum.

Estradiol regulates progesterone synthesis by the corpus luteum of several species. The molecular mechanism(s) of the action of estradiol is unknown, but appears to be independent of exogenous gonadotropins and tissue cAMP. A role for Ca2+ in steroidogenesis has been implicated, and we demonstrate here that estradiol alters Ca-specific protein phosphorylation in rat luteal cells. Corpora lutea obtained from rats hypophysectomized and hysterectomized on day 12 of pregnancy and subsequently treated for 3 days with vehicle or estradiol were assayed in vitro for Ca-specific phosphorylation. Estradiol elevated the content of a number of cytosolic proteins (mol wt X 10(-3), 58, 82, 100, 166, and 183); however, phosphorylation of the 100K protein alone appeared to be specifically enhanced by estradiol. In the presence of Ca2+, phosphorylation of the luteal 100K protein increased 2.33 +/- 0.2-fold in estradiol-treated vs. 1.42 +/- 0.2-fold in vehicle-treated rats (n = 6; P less than 0.05). Phosphorylation of 100K was inhibited by trifluoperazine and stimulated by calmodulin (CaM). Phosphopeptide maps revealed that the 100K luteal protein is identical to the cytosolic CaM-protein kinase III substrate, termed 100K, present in a number of tissues. Estradiol increased both 100K content and CaM-kinase III activity in luteal cells. Immunochemical analysis using antibodies prepared against pancreatic 100K revealed that estradiol treatment increased by at least 5 to 7-fold the luteal content of 100K. In addition, luteal cytosol of estradiol-treated rats enhanced phosphorylation of purified pancreatic 100K 3-fold, whereas that of vehicle-treated rats caused a 1.8-fold stimulation. The effects of estradiol on cytosolic proteins appear to be specific for 100K, since it does not after the activities of CaM-kinases I and II or cAMP-PK. In summary, results of this investigation demonstrate for the first time that estradiol increases the content of several proteins in the corpus luteum; estradiol enhances specifically the Ca-CaM-dependent phosphorylation of a 100K cytosolic protein; and the CaM-kinase III-100K substrate system is hormonally regulated.

Contrasting effects of prolactin on luteal and follicular steroidogenesis.

To determine whether prolactin affects both luteal and follicular production of testosterone and oestradiol, pseudopregnant rats, either intact or hypophysectomized on day 8, were injected daily between days 8 and 9 with 1.5 i.u. human chorionic gonadotrophin (hCG), 250 micrograms prolactin or a combination of both. Control rats were given vehicle. On day 9, blood was obtained from the ovarian vein and corpora lutea and follicles were isolated and incubated in vitro for 2 h. Administration of hCG to intact rats increased ovarian secretion of testosterone and oestradiol dramatically, but did not affect progesterone secretion. Hypophysectomy on day 8 of pseudopregnancy was followed by a drop in ovarian steroid secretion. Prolactin treatment of hypophysectomized rats markedly enhanced progesterone production but had no stimulatory effect on either testosterone or oestradiol. In contrast, hCG dramatically enhanced ovarian secretion of both testosterone and oestradiol without affecting progesterone secretion. Prolactin administered together with hCG antagonized the stimulation of both testosterone and oestradiol secretion by hCG, yet increased progesterone production. When the specific effects of hCG and prolactin administration on follicles and corpora lutea were studied separately, it was found that hCG treatment in vivo greatly stimulated testosterone and oestradiol production by both tissues in vitro. Since hCG only marginally affected aromatase activity in the follicle, had no effect on aromatase activity in luteal cells and did not increase progesterone synthesis, it appears that hCG acts to increase the formation of androgen substrate for oestradiol biosynthesis. Prolactin, administered with or without hCG, inhibited both basal and hCG-stimulated testosterone and oestradiol synthesis by the follicle. In sharp contrast to its inhibitory effect on follicular production of steroids, prolactin appears to be essential for LH stimulation of testosterone and oestradiol by the corpus luteum. In the absence of prolactin, luteal cells gradually ceased to respond to LH and decreased their output of testosterone and oestradiol. Prolactin administration to hypophysectomized rats did not affect luteal cell production of either steroid. However, corpora lutea of rats treated with prolactin responded to the hCG challenge with an increase in testosterone and oestradiol synthesis. In summary, results of this investigation demonstrate that prolactin affects follicular and luteal production of testosterone and oestradiol in opposite ways.(ABSTRACT TRUNCATED AT 400 WORDS)

Disparate Effects of Prostaglandins on Basal and Gonadotropin-Stimulated Progesterone Production by Luteal Cells Isolated from Rhesus Monkeys during the Menstrual Cycle and Pregnancy

To examine the direct effects of prostaglandins (PG) on corpus luteum function in primates, luteal cells isolated from rhesus monkeys were incubated in the presence of PGE2 or PGF2α, either alone, or in combination with gonadotropin (hCG) or dibutyryl cyclic AMP. Luteal cells were obtained at Days 16–18 (4–7 days after the preovulatory LH surge; midluteal phase cells) and Days 21–22 (8–10 days after the LH surge; late luteal phase cells) of the menstrual cycle and at 160–166 days of gestation (term pregnancy cells). Under control conditions, luteal cells at these stages produced similar amounts of progesterone in vitro. Progesterone production by cells from midluteal phase of the menstrual cycle was significantly (P<0.05) increased in the presence of 50 ng PCE2/ml, with a maximal response at 5000 ng/ml. PGF2α was 10-fold less potent than PGE2 in stimulating progesterone production by midluteal phase cells. In contrast, the progesterone synthetic activity of luteal cells from late luteal phase of the cycle and at term pregnancy was unaltered by the presence of 5–5000 ng/ml of either PGE2 or PGF2α. PG (5000 ng/ml) stimulation of progesterone secretion by midluteal phase cells was less (P<0.01) than that elicited by the presence of 1000 ng hCG/ml and the combination of hCG and either PGE2 or PGF2α produced a response no different from that by hCG alone. Although cells from late luteal phase of the cycle and term pregnancy responded to 1000 ng hCG/ml with enhanced (P<0.01) progesterone production, the combination of hCG and 5000 ng/ml of either PGE2 or PGF2α failed to increase progesterone secretion. However, neither PG prevented the stimulation of luteal cell progesterone production by 1 mM dibutyryl cyclic AMP. Thus, PG mimicked gonadotropin stimulation of luteal cell steroidogenesis at midluteal phase of the menstrual cycle, but antagonized the stimulatory effect of hCG on luteal cells isolated later in the cycle and at term pregnancy. PG may act prior to the elevation of cellular cyclic AMP to block the mechanism of hCG stimulation of progesterone production by luteal cells. The data suggest that PG may have a potential luteotropic or luteolytic effect on luteal cells of the rhesus monkey, depending upon the "age" of the corpus luteum.

Species variation in the distribution of cholinesterases in the ovary of the plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer.

Sections of ovary from plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer have been histochemically processed to demonstrate acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in nervous and non-nervous tissue. The effects of different reproductive states on enzyme activity were observed in some animals. AChE-containing nerves were sparse in rabbit and rat but plentiful in cat and roe deer. Nerves containing BuChE were not detectable in ferret or guinea-pig and were rare in cat. Species variations in the activity and type of enzyme were also found in non-neuronal tissues. Some blood vessels in the ovaries of guinea-pig and viscacha contained AChE. No other species showed a reaction for AChE in non-neuronal stromal tissue but BuChE was present at this site in all animals except rat. Granulosa cells reacted for AChE only in cat and rabbit while luteal cells were reactive in cat, rabbit and roe deer. Some BuChE activity was present in granulosa and or luteal cells in all species except roe deer. In rat, BuChE activity in luteal cells increased during pregnancy and the early phase of pseudopregnancy. The difficulty of assigning a function to ovarian cholinesterases is discussed.

Gonadotropin-Sensitive Progesterone Production by Rhesus Monkey Luteal Cells in Vitro: A Function of Age of the Corpus Luteum During the Menstrual Cycle

Progesterone production in vitro, in the presence and absence of exogenous gonadotropin, was examined in suspensions of luteal cells, isolated by collagenase digestion of rhesus monkeys corpus luteum at various stages of the menstrual cycle. Cells isolated during mid-luteal phase (days 15-19) of the cycle secreted progesterone for up to 6 h in vitro. Mid-luteal phase cells were responsive to physiologic concentrations of human chorionic gonadotropin (hCG), with progesterone production significantly (P less than 0.05) enhanced by as little as 0.1 ng hCG/ml. Maximal stimulation was obtained with 100 ng hCG/ml. Both macaque chorionic gonadotropin (mCG) and human luteinizing hormone (hLH) significantly (P less than 0.01) increased progesterone production, while human follicle stimulating hormone (hFSH) did not. Under control conditions, in the presence of nutrient medium alone (no exogenous gonadotropin), the progesterone synthetic activity of mid-luteal phase cells was significantly (P less than 0.01) greater than that of cells from late luteal phase (days 22-28) of the cycle. Moreover, progesterone production by mid-luteal phase cells was consistently stimulated (P less than 0.01) by the presence of 100 ng hCG/ml, whereas late luteal phase cells were less sensitive or unresponsive to exogenous gonadotropin. The progesterone synthetic activity of luteal cells in vitro correlated positively with both the wet weight of the excised corpus luteum (r = 0.82, P less than 0.01) and the peripheral serum progesterone concentration immediately preceding luteectomy (r = 0.66, P less than 0.01). These findings suggest that freshly isolated luteal cells reflect the functional capability of the corpus luteum in vivo. It is apparent that the age of the rhesus monkey corpus luteum of the non-fertile menstrual cycle is an important factor governing luteal cell progesterone synthetic capability and luteal cell responsiveness to gonadotropin in vitro.