Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.     

Prostaglandins and in vitro ovarian progestin biosynthesis

Prostaglandin F_2α (PGF_2α) did not alter the $\text{in vitro}$ biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF_2α did not affect the incorporation of acetate-1-¹⁴C into progestins. PGF_1α, 15-keto PGF_2α, and PGE₁ did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE₂ inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF_2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF_2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF_2α (10 μg/ml) did not stimulate the $\text{in vitro}$ biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Role of prostaglandin F2α in modulation of LH-stimulated steroidogenesis in vitro in different types of rat and ewe corpora lutea

An inhibitory effect of PGF_2α at a dose of 7 × 10^?7 M on LH stimulated synthesis of progesterone was observed $\text{in vitro}$ after incubation of pseudopregnant rat ovaries for a period of 2 hours. A similar effect was seen with cyclic and gestant ewe corpora lutea at the same dose of PGF_2α. This effect was observed both in the secretion of progesterone and on the amount of progesterone present in the tissue. This inhibitory effect of PGF_2α on LH stimulated progesterone synthesis may explain the modification in the time course for gonadotropin action in luteal tissue at high and low doses.     

The role of microfilaments and of microtubules in taurocholate uptake by isolated rat liver cells

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [¹⁴C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging $\text{0.87 ±}\text{SD}\text{0.05}\text{nmol}\text{· s}{}^{\mathrm{?1}}\text{· 10}{}^{\mathrm{?6}}\text{cells}$ and $\text{10.9 ± 1.8 μ}\text{M}$, respectively. Cytochalasin B ($\text{4.2–420 μ}\text{M}$) inhibited taurocholate uptake in a competitive fashion; $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{33 ± 7 μ}\text{M}$. At concentrations above 100 μM the compound decreased ³⁶Cl membrane potential and intracellular K⁺ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1–1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{0.47 ± 0.07}\text{mM}$. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.     

The effects of cytochalasin B and vinblastine on movement of cholesterol in rat adrenal glands.

Vinblastine (antimicrotubular agent) and cytochalasin B (antimicrofilament agent) block the build up of adrenal mitochondrial cholesterol seen in the presence of AMG. ACTH stimulated steroidogenesis is inhibited $\text{in vivo}$ by both agents via a reduction in the transfer of intra-adrenal cholesterol to adrenal mitochondria, resulting in a decrease in the synthesis of adrenal steroids. Both inhibitors also decrease ACTH stimulated formation of cholesterol cytochrome P450_SCC complex in adrenal mitochondria, as determined by difference spectroscopy. The effects of these inhibitors contrast with the actions of protein synthesis inhibitors which decrease cholesterol binding to P450_SCC while increasing mitochondrial cholesterol content.     

Conversion of cholesterol to progesterone by turtle corpus luteum

Homogenates of corpora lutea from the snapping turtle, $\text{Chelydra}$$\text{serpentina serpentina}$ were capable of converting small (< 1%) amounts of cholesterol-³H to progesterone-³H. There was about twice as much steroidogenic activity in corpora lutea taken from animals still carrying oviducal eggs as in those from animals that had laid their eggs. However, the latter showed up to an 80% increase in conversion of cholesterol to progesterone when turtle pituitary homogenate was incubated along with the luteal homogenate. Approximately 4 and 9 mg/g of free and esterified sterol, respectively, were found in the turtle corpus luteum. These results suggest that the corpus luteum of the oviparous turtle functions as a steroidogenic organ.     

In. vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: Progesterone production by dispersed luteal cells

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term $\text{in vitro}$ studies of progesterone production by suspensions of collagenase-dispersed luteal cells in the presence and absence of exogenous gonadotropin (human chortonic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the incubation medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion above basal level ($\text{63.7 ± 13.1}\text{versus}\text{24.7 ± 5.5}\text{ng progesterone}\text{/}\text{ml}\text{/5 × 10}{}^4\text{cells}\text{/ 3}\text{hr}\text{,}\text{X}\text{± S.E.,}\text{n}\text{= 6;}\text{p}\text{< 0.05}$). However, luteal cells from early pregnancy (23–26 days after fertilization) secreted significantly less progesterone than cells of the non-fertile menstrual cycle (3.6 ± 2.4 versus 24.7 ± 5.5 ng/ml/5 × 10⁴ cells/3 hr, n = 3; p < 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108–118 days gestation) luteal cells exhibited partially renewed function, and near the time of parturition (163–166 days gestation) basal and HCG-stimulated progesterone secretion (30.2 ± 5.6 and 63.0 ± 13.0 ng/ml/5 × 10⁴ cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when steroidogenic activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitive steroidogenesis similar to that of the corpus luteum of the menstrual cycle.     

The effects of prostacyclin (PGI2) and 6-keto-PGF1α on bovine plasma progesterone and LH concentrations

Injections of 1 mg PGI₂ directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI₂-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF_1α were without effect on plasma progesterone levels. The luteotrophic effect of PGI₂ was not due to alterations in circulating LH concentrations. An $\text{in vitro}$ experiment assessed the effects of either PGI₂ alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 μg PGI₂, 10 μg PGI₂, and 10 μg PGI₂ plus 5 ng LH averaged 99 ± 42, 353 ± 70, 152 ± 35, 252 ± 45, and 287 ± 66 ng/ml (n=4), respectively. Thus PGI₂ has luteotrophic effects on the bovine CL both $\text{in vivo}$ and $\text{in vitro}$.     

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-³H]cytochalasin B and transport of $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-[}{}^{14}\text{C]glucose}$ into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a K_D of 2.86 · 10^?7 M, while the low-affinity site had a K_D of 1.13 · 10^?5M. Sugar transport was monitored by $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-}\text{glucose}$ uptake and it was found that cytochalasin B (10^?5M) drastically inhibited transport. However, D-glucose (10^?5M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.     

Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

Divergent effects of prolactin on 4-ene-5α-reductase activity and production of C19-steroids from progesterone in immature rat ovary

Female rats of the Sprague-Dawley strain were used. Two pituitaries from 9-week old rats were grafted in both kidneys of 21-day old rat to induce hyperprolactinemia. All rats with or without pituitary isografts were hypophysectomized on day 26. Starting from day 29, the rats in groups of 8–11 were injected daily with 5 μg NIH-LH-S19 or saline for 3 days. Ovarian homogenates from these rats on day 32 were incubated with [¹⁴C]4-androstene-3, 17-dione or [³H]progesterone and steroid metabolism was estimated. In the hypophysectomized rat ovary, the 5α-reductase activity was stimulated significantly by LH. Although pituitary isograft alone had no stimulative effect on 5α-reductase activity of the hypophysectomized rat ovary, concomitant treatment with LH and pituitary isograft (prolactin) had an additive effect. Formation of the sum of C₁₉-steroids from progesterone in the hypophysectomized rat ovary was stimulated markedly by LH but reduced slightly by prolactin. The LH-induced production of C₁₉-steroids from progesterone was inhibited markedly by prolactin.These results indicate that prolactin treatment inhibits basal and LH-induced production of C₁₉-steroids from progesterone but stimulates LH-induced 4-ene-5α-reductase activity in immature rat ovary.     

Implication of microtubules and microfilaments in the response of the ovarian adenylate cyclase-cyclic AMP system to gonadotropins and prostaglandin E2.

Addition of anti-actin serum or cytochalasin B (3 μg/ml) to the medium abolished the stimulatory effect of LH and of choleragen, and inhibited the action of FSH, but not of PGE₂, on cyclic AMP production in cultured rat Graafian follicles. Colchicine and anti-sera to BSA, tubulin or smooth-muscle myosin, as well as anti-actin serum absorbed with actin, had no effect on the follicular response to LH, but anti-tubulin serum and colchicine inhibited the response to FSH and PGE₂. The inhibitory effect of cytochalasin B on LH-action was fully reversed 24 h after transfer of the follicles to drug-free medium. Neither anti-actin serum nor cytochalasin B had any effect on the binding of ¹²⁵I-hCG by the follicular cell membrane. The results suggest that microfilaments, but not microtubules, are intimately involved in the process of LH- and choleragen-stimulated ovarian adenylate cyclase activity. By contrast, the action of PGE₂ is dependent on microtubule assembly, while the action of FSH seems to depend on both these components of the cytoskeleton.     

Role of gangliosides in gonadotropin and cholera enterotoxin stimulated steroidogenesis in isolated rat ovarian cells

Ovarian cells isolated from 26 day old rats responded to hCG (10 ng/ml) and cholera enterotoxin (100 ng/ml) $\text{in vitro}$ with a forty-five to fifty-fold increase in progesterone production. Both cholera enterotoxin and hCG-stimulated progesterone response was accompanied by a lag period. The duration of the lag period in the production of the progesterone depended on the concentration of gonadotropin or cholera enterotoxin, and with maximally stimulating dose it was 20–30 minutes. Addition of highly purified mixed gangliosides to the incubation medium abolished the stimulatory effect of cholera enterotoxin on progesterone response. In contrast, under identical experimental conditions, ganglioside addition produced no effect on progesterone response elicited by hCG or LH. Similarly mixed gangliosides did not prevent the specific binding of [¹²⁵I]hCG to the ovarian cells or to the membranes isolated from the ovary. In addition preincubation of [¹²⁵I]hCG with ganglioside did not alter the subsequent binding of the hormone to the ovarian cell surface receptor. These findings suggest that gangliosides are not involved in the hormone receptor interactions and subsequent receptor mediated physiological response.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.     

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.     

Effect of PGF2α on progesterone secretion and adenylate cyclase activity in ovine luteal tissue

Ovine luteal slices were used to study the effects of prostaglandins (PG) F₂α on luteinizing hormone (LH)-stimulated secretion of progesterone and adenylate cyclase activity. The accumulation of progesterone in incubation medium and adenylate cyclase activity was similar after incubation of luteal slices with Medium 199 alone or Medium 199 containing PGF₂α (250 ng/ml) for 3 hr. Addition of luteinizing hormone (LH; 100 ng/ml) resulted in a 2–3 fold increase in both the rate of progesterone accumulation and adenylate eyclase activity by 3 hr. When luteal slices were incubated in the presence of both LH and PGF₂α the rates of progesterone accumulation and adenylate cyclase activity were identical to those in flasks containing LH alone after 1 hr; however, after 3 hr both LH stimulated progesterone accumulation and adenylate cyclase activity were inhibited to levels similar to those observed in control slices.In a second experiment, after 60–120 min of exposure to PGF₂α the rate of progesterone accumulation in the medium was not different from that in untreated control slices. In addition, after this experiment the luteal slices were homogenized and the basal, sodium fluoride, LH, isoproterenol (ISO) and PGE₂ sensitive adenylate cyclase activities were determined to evaluate the hormonal specificity of the negative effect of the pretreatment with PGF₂α. Both LH and ISO stimulated adenylate cyclase activities were reduced after PGF₂α pretreatment. However, fluoride ion stimulated adenylate cyclase activity was not significantly effected by PGF₂α pretreatment and PGE₂ sensitive adenylate cyclase was effected only slightly.     

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

Lipid and arachidonic acid accumulation in naturally regressing porcine corpora lutea

Patterns of luteal lipid and arachidonic acid accumulation were examined in relation to luteal progesterone and prostaglandin F synthesis in 30 sows and gilts between days 8 and 18 of the estrous cycle. Net $\text{in vitro}$ release of progesterone from luteal tissue declined from 722 ng/100 mg tissue at day 8 to 81 ng/100 mg tissue at day 18. Although statistical significance was not present, net prostaglandin F release increased slightly from 8.6 to 13.9 ng/100 mg tissue. Luteal free cholesterol, esterified cholesterol, and free fatty acid contents did not change between days 8 and 18 whereas triglycerides accumulated rapidly between days 14 and 18 of the estrous cycle. Phospholipids increased between days 8 and 12, plateaued at 20.2 mg/g between days 14 and 16, and decreased to 15.4 mg/g on day 18. Between days 12 and 18, arachidonic acid increased from 19.4 to 34.8% in cholesterol esters, from 10.1 to 22.5% in triglycerides, and from 12.3 to 27.2% in luteal free fatty acids. Arachidonic acid in luteal phospholipids increased from 21.3 to 25.1% between days 14 and 16 of the estrous cycle. Luteal regression was associated with conservation of arachidonic acid. Based on blood plasma lipid fatty acid compositions, the corpus luteum elongated and desaturated essential fatty acids. Within porcine corpora lutea, calculated free arachidonic acid content was adequate for maintenance of prostaglandin synthesis.     

Effect of cytochalasin B and colchicine on the stimulation of -amylase release from rat parotid tissue slices

A role for microfilaments and microtubules in the secretion of α-amylase is indicated since cytochalasin B and colchicine inhibited the stimulation of α-amylase release by epinephrine (30 or 15 μM) but only cytochalasin B inhibited the stimulation by N⁶, O^2′ dibutyryl adenosine 3′,5′monophosphate (1.0 mM). It was necessary to incubate the parotid tissue slices in the presence of cytochalasin B (1 hr.) or colchicine (4 hrs.) before adding the agonist in order to see the inhibitory effects.