Increase In Progesterone Synthesis Research Articles

High concentrations of progesterone inhibit the expression of genes related to steroid metabolism in MA-10 Leydig cells

Leydig cells are the main testosterone-producing cells in males. During androgen synthesis, cholesterol enters the mitochondria via the STAR protein and is converted into pregnenolone by the CYP11A1 enzyme. This steroid is then exported from the mitochondria to be metabolized to progesterone by the HSD3B1 enzyme in the endoplasmic reticulum. In this study, we used 3′Tag-RNA-Seq to identify progesterone-regulated genes in MA-10 Leydig cells. Our results indicate that high concentrations of progesterone (30 μM) are involved in a negative feedback loop that inhibits cAMP/PKA-dependent activation of Star and Cyp11a1 expression and participate in cAMP/PKA-dependent down-regulation of genes related to the metabolism of steroid hormones. Linked to activation of the MAPK signaling pathway, endoplasmic reticulum stress and apoptosis, most of the genes encoding bZIP transcription factors are upregulated by progesterone in MA-10 Leydig cells. However, only DDIT3 protein levels are increased in response to progesterone in MA-10 Leydig cells. Like normal Leydig cells, MA-10 cells very weakly express the classical nuclear receptor for progesterone, suggesting that gene regulation by progesterone is rather mediated by one of the non-classical membrane receptors for progesterone However, current findings suggest that the inhibitory effect of progesterone on STAR protein increase in response to forskolin is not dependent on PGRMC1/2 or PAQR9. Furthermore, the increase in progesterone synthesis in response to activation of the cAMP/PKA pathway is rather inhibited by siRNA-mediated knockdown of PAQR9. Overall, this study shows that progesterone produced by Leydig cells participates in the regulation of steroidogenesis through autocrine action involving negative feedback upon activation of the cAMP/PKA pathway.

Ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation mediates female reproductive dysfunction induced by cold exposure

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to β3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

MiR-140-3p promotes follicle granulosa cell proliferation and steroid hormone synthesis via targeting AMH in chickens.

Granulosa cells (GCs) are the ovary's most critical cells since they undergo cell differentiation and hormone synthesis changes closely associated with follicle development. While micro RNA 140-3p (miRNA-140-3p) has an apparent cell signaling role, particularly in cell proliferation, its biological role in chicken ovarian follicle growth and development remains elusive. This study explored miR-140-3p's effects on chicken GC proliferation and steroid hormone synthesis. MiR-140-3p dramatically increased GC proliferation, prevented apoptosis, increased progesterone synthesis, and enhanced gene expression related to steroid hormone synthesis. In addition, the anti-Müllerian hormone (AMH) gene was identified as a direct miR-140-3p target. MiR-140-3p abundance correlated negatively with AMH mRNA and protein levels in GCs. Our findings show that miR-140-3p influences chicken GC proliferation and steroid hormone synthesis by suppressing AMH expression.

ODP266 Deficiency of Calcitriol and Sex Hormones, Which Leads to a Severe Course of Psoriasis

Abstract Materials and methods The study group consisted of 17 patients with psoriasis from 19 to 76 years. All patients underwent studies to determine the body's vitamin D supply by its metabolite calcidiol 25(OH)D by enzyme immunoassay and to determine the level of steroid hormones by the steroid profile of saliva by mass spectrometry: progesterone, testosterone, estradiol. Results Vitamin D insufficiency was detected in 9 patients (n=9, 52.9%), the average level was 24.3±0.96 ng/ml. Vitamin D deficiency was observed in 3 patients (n=3, 17.7%), the average level was 16.6±1.634 ng/ml. Adequate vitamin D levels was detected in 5 patients (n=5, 29.4%), the average level was 34.56±1.955 ng/ml. In all patients (n=17, 100%), psoriasis was in a progressive stage, PASI more than 20 points – severe psoriasis. When evaluating the steroid profile of saliva in 7 patients (n=7, 41.2%) the level of progesterone was below the reference values (reference values 80-180 pg/ml), average level 56.86±6.53 pg/ml. In 11 patients (n=11, 64.7%), including 7 patients with progesterone deficiency plus 4 patients whose progesterone levels are within the reference values, have the level of testosterone was below the reference values (reference values from 114.8 pg/ml) the average level 51.93±9.5 pg/ml. Discussion Considering steroidogenesis [1], even though only 4 (n=4, 23.5%) patients had estradiol levels below reference values (reference values 4.17-9.28 pg/ml), we conclude that estradiol deficiency was observed in at least 11 patients (n=11, 64.7%). Also, knowing the effect of vitamin D on steroidogenesis (increases the expression of the 3b-HSD gene, thereby increasing progesterone synthesis [2]), thus we already know that if a patient is vitamin D insufficient or deficient, even when within the reference values, progesterone levels will not be sufficient for this individual, and therefore there will be testosterone and estradiol deficiency. It follows that another 8 patients with vitamin D insufficiency or deficiency will have progesterone deficiency, that is, out of 17 patients, 15 (n=15, 88.2%) have progesterone deficiency and, therefore, testosterone and estradiol deficiency. In enzyme immunoassay vitamin D determination, a cross-reaction occurs with another metabolite - 24.25(OH)D, whose concentration is up to 20% of the total calcidiol concentration. Therefore, it is necessary to subtract up to 20% from the data obtained in order to determine the true level of 25(OH)D [3], which means that all patients have vitamin D deficiency. Estrogens enhance the synthesis and effects of 1,25(OH)D, through an increase in the expression of the CYP27B1 (1α-hydroxylase) gene and VDR [4]. Conclusion The sufficient intake of vitamin D and a sufficient level of calcidiol, the intensity of the synthesis of the hormonal form of vitamin D and the realization of its effects depend on several factors, which include a sufficient level of sex hormones. Presentation: No date and time listed

Response to “Comment on ‘Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors’”

Response to “Comment on ‘Application of an <i>in Vitro</i> Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors’”

A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

Inhibition of mitochondrial fission and protein kinase R improves progesterone in placental stress.

Placenta synthesizes hormones that play a vital role in adapting maternal physiology and supporting fetal growth. This study aimed to explore the link between progesterone, a key steroid hormone produced by placenta, and mitochondrial fission and protein kinase R through the use of chemical inhibition in trophoblasts subjected to endotoxin lipopolysaccharide and double-stranded RNA analog polyinosinic:polycytidylic acid stress. Expressions of protein kinase R, dynamin-related protein 1, mitochondrial fission protein 1, and heat shock protein 60 were determined by applying lipopolysaccharide and polyinosinic:polycytidylic acid to BeWo trophoblast cells. Next, cells were treated with protein kinase R inhibitor 2-aminopurine and mitochondrial division inhibitor 1 to examine changes in progesterone levels and expression levels of proteins and mRNAs involved in progesterone biosynthesis. Last, effect of 2-aminopurine on mitochondrial fission was determined by immunoblotting and quantitative PCR (qPCR). Mitochondrial structural changes were also examined by transmission electron microscopy. Lipopolysaccharide and polyinosinic:polycytidylic acid stimulation induced mitochondrial fission and activated protein kinase R but decreased heat shock protein 60 levels and progesterone synthesis. Chemical inhibition of mitochondrial fission elevated progesterone synthesis and protein and mRNA levels of genes involved in progesterone biosynthesis. Inhibition of protein kinase R with 2-aminopurine prevented lipopolysaccharide and polyinosinic:polycytidylic acid induced mitochondrial fission and increased progesterone biosynthesis. Use of chemical inhibitors to treat placental stress caused by pathogens has potential to stabilize the production of progesterone. The study reveals that inhibiting mitochondrial fragmentation and reducing activity of stress kinase protein kinase R in syncytiotrophoblasts leads to an increase in progesterone synthesis when exposed to lipopolysaccharide and polyinosinic:polycytidylic acid.

Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors.

Background:Established breast cancer risk factors, such as hormone replacement therapy and reproductive history, are thought to act by increasing estrogen and progesterone (P4) activity.Objective:We aimed to use in vitro screening data to identify chemicals that increase the synthesis of estradiol (E2) or P4 and evaluate potential risks.Method:Using data from a high-throughput (HT) in vitro steroidogenesis assay developed for the U.S. Environmental Protection Agency (EPA) ToxCast program, we identified chemicals that increased estradiol (E2-up) or progesterone (P4-up) in human H295R adrenocortical carcinoma cells. We prioritized chemicals by their activity. We compiled in vivo studies and assessments about carcinogenicity and reproductive/developmental (repro/dev) toxicity. We identified exposure sources and predicted intakes from the U.S. EPA’s ExpoCast.Results:We found 296 chemicals increased E2 (182) or P4 (185), with 71 chemicals increasing both. In vivo data often showed effects consistent with this mechanism. Of the E2- and P4-up chemicals, about 30% were likely repro/dev toxicants or carcinogens, whereas only 5–13% were classified as unlikely. However, most of the chemicals had insufficient in vivo data to evaluate their effects. Of 45 chemicals associated with mammary gland effects, and also tested in the H294R assay, 29 increased E2 or P4, including the well-known mammary carcinogen 7,12-dimethylbenz(a)anthracene. E2- and P4-up chemicals include pesticides, consumer product ingredients, food additives, and drinking water contaminants.Discussion:The U.S. EPA’s in vitro screening data identified several hundred chemicals that should be considered as potential risk factors for breast cancer because they increased E2 or P4 synthesis. In vitro data is a helpful addition to current toxicity assessments, which are not sensitive to mammary gland effects. Relevant effects on the mammary gland are often not noticed or are dismissed, including for 2,4-dichlorophenol and cyfluthrin. Fifty-three active E2-up and 59 active P4-up chemicals that are in consumer products, food, pesticides, or drugs have not been evaluated for carcinogenic potential and are priorities for study and exposure reduction. https://doi.org/10.1289/EHP8608

Progesterone Actions During Central Nervous System Development.

Although progesterone is a steroid hormone mainly associated with female reproductive functions, such as uterine receptivity and maintenance of pregnancy, accumulating data have shown its physiological actions to extend to several non-reproductive functions in the central nervous system (CNS) both in males and females. In fact, progesterone is de novo synthesized in specific brain regions by neurons and glial cells and is involved in the regulation of various molecular and cellular processes underlying myelination, neuroprotection, neuromodulation, learning and memory, and mood. Furthermore, progesterone has been reported to be implicated in critical developmental events, such as cell differentiation and neural circuits formation. This view is supported by the increase in progesterone synthesis observed during pregnancy in both the placenta and the fetal brain. In the present review, we will focus on progesterone actions during CNS development.

Relationship Between Dietary Fatty Acids and Reproductive Functions in Dairy Cattle

Selection of dairy cattle for higher milk yield, without considering important non-production traits, has decreased reproductive efficiency. Thus, low reproductive performance is a major problem in high yielding dairy cattle. Previous studies showed that dietary manipulation to improve fertility holds much promise and dietary fats have positive effects on reproductive functions in high yielding dairy cattle. Positive effects of fats on reproductive performance due to the fatty acids, which are the precursors of progesterone and prostaglandins. Progesterone and prostaglandins hormones are most important factors that play a role on the control of reproductive functions. The amount of linoleic, linolenic and arachidonic fattty acids in ration can be increase or decrease progesterone and prostaglandins synthesis especially PGF2α from ovary and uterus, respectively. Also fatty acids can be influence follicular development, ovulation, embryonic implantation and maternal recognition of pregnancy. This review focuses on the relationships between dietary fatty acids and reproductive functions such as hormone profiles, ovarian function and follicular development, oocyte quality, embryo development, embryonic implantation and maternal recognition of pregnancy in dairy cattle.

Kisspeptin regulates ovarian steroidogenesis during delayed embryonic development in the fruit bat, Cynopterus sphinx.

Cynopterus sphinx, a fruit bat, undergoes delayed embryonic development during the winter months, a period that corresponds to low levels of progesterone and estradiol synthesis by the ovary. Kisspeptins (KPs) are a group of neuropeptide hormones that act via G-protein coupled receptor 54 (GPR54) to stimulate hypothalamic secretion of Gonadotropin-releasing hormone, thereby regulating ovarian steroidogenesis, folliculogenesis, and ovulation. GPR54 is also expressed in the ovary, suggesting a direct role for KPs in ovarian steroidogenesis. The aim of present study was to determine if a low serum level of KP is responsible for reduced progesterone and estradiol levels during the period of delayed embryonic development in C. sphinx. Indeed, low serum KP abundance corresponded to reduced expression of GPR54 in ovarian luteal cells during the period of delayed development compared to normal development. In vitro and in vivo treatment with KP increased GPR54 abundance, via Extracellular signal regulated kinase and its downstream mediators, leading to increased progesterone synthesis in the ovary during delayed embryonic development. KP treatment also increased cholesterol uptake and elevated expression of Luteinizing hormone receptor and Steroid acute regulatory protein in the ovary, suggesting that elevation in circulating KP during delayed embryonic development may reactivate luteal activity. KPs may also enhance cell survival (BCL-2, reduced Caspase 3 activity) and angiogenesis (Vascular endothelium growth factor) during this period. The findings of this study thus demonstrate a regulatory role for KPs in the maintenance of luteal steroidogenesis during pregnancy in C. sphinx.

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast

BackgroundSTARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. MethodsProgesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. ResultsSTARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. ConclusionMetalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. General significanceMitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.

Membrane potential regulates mitochondrial ATP-diphosphohydrolase activity but is not involved in progesterone biosynthesis in human syncytiotrophoblast cells

ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and progesterone synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased progesterone synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased progesterone synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.

Elevated non-esterified fatty acid concentrations during in vitro murine follicle growth alter follicular physiology and reduce oocyte developmental competence

To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro. Experimental study. In vitro culture setting. Female and male 13-day old, B6CBAF1 mice of proven fertility were sacrificed for harvesting ovaries and epididymal sperm, respectively. Early secondary murine follicles were cultured in vitro in the presence of NEFAs until the antral stage (12 days). Treatments consisted of one or a mixture of NEFAs (stearic acid [SA], palmitic acid [PA], oleic acid [OA]) in physiological (basal) or pathological (high SA, high OA, high NEFA) concentrations. Follicular development; follicle and oocyte diameters; secretion of progesterone, estradiol, and inhibin B; and luteinized granulosa cell gene expression patterns were investigated. Oocytes from NEFA-exposed follicles were fertilized in vitro, and presumptive zygotes were cultured until the blastocyst stage. Exposure to high SA reduced follicle diameters and day-12 antrum formation. Elevated NEFA concentrations changed luteinized granulosa cell messenger-ribonucleic acid abundance of genes related to energy/fatty acid/steroid metabolism, apoptosis, and oxidative stress. High NEFA and high SA treatments increased progesterone synthesis, compared with high OA follicles. Oocyte developmental competence was substantially reduced in oocytes retrieved from high OA-, high SA-, and high NEFA-exposed follicles compared with basal-treated follicles. This study showed, for the first time, that lipolysis-linked, elevated NEFA concentrations can potentially impair fertility, by altering follicular physiology and reducing oocyte developmental competence.

Neurosteroids, trigger of the LH surge

Recent experiments from our laboratory are consistent with the idea that hypothalamic astrocytes are critical components of the central nervous system (CNS) mediated estrogen positive feedback mechanism. The “astrocrine hypothesis” maintains that ovarian estradiol rapidly increases free cytoplasmic calcium concentrations ([Ca2+]i) that facilitate progesterone synthesis in astrocytes. This hypothalamic neuroprogesterone along with the elevated estrogen from the ovaries allows for the surge release of gonadotropin-releasing hormone (GnRH) that triggers the pituitary luteinizing hormone (LH) surge. A narrow range of estradiol stimulated progesterone production supports an “off-on-off” mechanism regulating the transition from estrogen negative feedback to estrogen positive feedback, and back again. The rapidity of the [Ca2+]i response and progesterone synthesis support a non-genomic, membrane-initiated signaling mechanism. In hypothalamic astrocytes, membrane-associated estrogen receptors (mERs) signal through transactivation of the metabotropic glutamate receptor type 1a (mGluR1a), implying that astrocytic function is influenced by surrounding glutamatergic nerve terminals. Although other putative mERs, such as mERβ, STX-activated mER-Gαq, and G protein-coupled receptor 30 (GPR30), are present and participate in membrane-mediated signaling, their influence in reproduction is still obscure since female reproduction be it estrogen positive feedback or lordosis behavior requires mERα. The astrocrine hypothesis is also consistent with the well-known sexual dimorphism of estrogen positive feedback. In rodents, only post-pubertal females exhibit this positive feedback. Hypothalamic astrocytes cultured from females, but not males, responded to estradiol by increasing progesterone synthesis. Estrogen autoregulates its own signaling by regulating levels of mERα in the plasma membrane of female astrocytes. In male astrocytes, the estradiol-induced increase in mERα was attenuated, suggesting that membrane-initiated estradiol signaling (MIES) would also be blunted. Indeed, estradiol induced [Ca2+]i release in male astrocytes, but not to levels required to stimulate progesterone synthesis. Investigation of this sexual differentiation was performed using hypothalamic astrocytes from post-pubertal four core genotype (FCG) mice. In this model, genetic sex is uncoupled from gonadal sex. We demonstrated that animals that developed testes (XYM and XXM) lacked estrogen positive feedback, strongly suggesting that the sexual differentiation of progesterone synthesis is driven by the sex steroid environment during early development.This article is part of a Special Issue entitled ‘Neurosteroids’.

English

Various nutritional tools have been used to improve the productive and reproductive performance of animals, among which difference between certain minerals, called dietary cation anion difference (DCAD) plays a pivotal role. Low or negative DCAD diets reduce blood pH and HCO3- and animal becomes acidotic. This improves Ca absorption from the intestinal tract. It also induces mobilization of Ca from bones which improves Ca status of the animal, thus preventing the occurrence of milk fever at the time of parturition. This may increase milk production and health in subsequent lactation. However, animals fed high DCAD diets before parturition may suffer from milk fever. Milk fever affected animals have increased plasma cortisol level that causes immunosuppression at calving. It is also positively associated with other problems like retained placenta, mastitis and udder edema. On the other hand, feeding high DCAD diet results in increased ruminal pH which is pre-requisite for optimal microbial activity as well as improving the feed intake of the animal. Improved dry matter intake (DMI) is positively correlated with milk yield by providing precursors for various milk constituents. High DCAD diet results in increased milk fat percent due to shifting of ruminal volatile fatty acid production towards acetic acid and butyric acid. It also improves energy balance of the animal which causes increased blood flow towards ovaries and increased progesterone synthesis and follicular development due to positive association between energy balance and postpartum ovulation, which leads to improved reproductive performance of the animal. While feeding low DCAD diet reduces feed intake which causes negative energy balance in early lactating animals that lessens conception rate and increases services per conception. In conclusion, feeding low DCAD diets prepartum prevents the occurrence of milk fever via improving Ca status while feeding high DCAD diets results in improved productive and reproductive performance in lactating animals. Key words: Feed intake, milk yield, reproductive performance, hypocalcaemia, dietary cation anion difference.

HCG-Induced Down-Regulation of PPARγ and Liver X Receptors Promotes Periovulatory Progesterone Synthesis by Macaque Granulosa Cells

An ovulatory stimulus induces the rapid and dramatic increase in progesterone synthesis by the primate ovarian follicle. However, little is known about the early events leading to the shift from estrogen to progesterone production. Because steroidogenesis represents an aspect of cholesterol metabolism, it was hypothesized that transcription factors regulating cholesterol balance would be among the earliest to change in response to an ovulatory stimulus. Granulosa cells were isolated from rhesus monkey follicles following controlled ovarian stimulation protocols before or up to 24 hr after an ovulatory human chorionic gonadotropin (hCG) bolus. The peroxisome proliferator-activated receptor-γ (PPARG) and the liver X receptors [nuclear receptor (NR)1H2, NR1H3] decreased within 3 hr of hCG, as did the reverse cholesterol transporters ATP-binding cassette (ABC)A1 and ABCG1. Treatment of granulosa cells isolated before an ovulatory stimulus with hCG and rosiglitizone resulted in an increase in NR1H3 and ABCG1, and decreased steroidogenic acute regulatory (STAR) protein and scavenger receptor-BI (SCARB1). A liver X receptor agonist attenuated hCG-induced progesterone synthesis in vitro and increased the expression of ABCA1 and ABCG1, and suppressed STAR, P450 side-chain cleavage A1, hydroxysteroid dehydrogenase 3B, and SCARB1. These data suggest that an initial action of LH/CG on the primate preovulatory follicle is to rapidly reduce the expression of PPARG, resulting in reduced NR1H3 with the consequence shifting the balance from cholesterol efflux via ABCA1 and ABCG1 to cholesterol uptake (SCARB1) and metabolism (STAR, P450 side-chain cleavage A1, hydroxysteroid dehydrogenase 3B). That the regulation of PPARG and the liver X receptors occurs within 3 hr strongly indicates that early events in the primate luteinizing follicle are critical to successful ovulation and luteal formation.

Melatonin regulates delayed embryonic development in the short-nosed fruit bat, Cynopterus sphinx

The aim of the present study was to evaluate the seasonal variation in serum melatonin levels and their relationship to the changes in the serum progesterone level, ovarian steroidogenesis, and embryonic development during two successive pregnancies of Cynopterus sphinx. Circulating melatonin concentrations showed two peaks; one coincided with the period of low progesterone synthesis and delayed embryonic development, whereas the second peak coincided with regressing corpus luteum. This finding suggests that increased serum melatonin level during November-December may be responsible for delayed embryonic development by suppressing progesterone synthesis. The study showed increased melatonin receptors (MTNR1A and MTNR1B) in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. The in vitro study showed that a high dose of melatonin suppressed progesterone synthesis, whereas a lower dose of melatonin increased progesterone synthesis by the ovary. The effects of melatonin on ovarian steroidogenesis are mediated through changes in the expression of peripheral-type benzodiazepine receptor, P450 side chain cleavage enzyme, and LH receptor proteins. This study further showed a suppressive impact of melatonin on the progesterone receptor (PGR) in the utero-embryonic unit; this effect might contribute to delayed embryonic development in C. sphinx. The results of the present study thus suggest that a high circulating melatonin level has a dual contribution in retarding embryonic development in C. sphinx by impairing progesterone synthesis as well as by inhibiting progesterone action by reducing expression of PGR in the utero-embryonic unit.

Involvement of peroxisome proliferator-activated receptor γ in gonadal steroidogenesis and steroidogenic acute regulatory protein expression

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to the PPAR family of nuclear transcription factors whose ligands, such as eicosanoids, fatty acids and prostaglandins, are known to affect gonadal function. Although several of these enhance the expression of the steroidogenic acute regulatory protein (STAR) and steroid production, the role of PPARgamma in regulating STAR-mediated steroidogenesis remains unclear. In the present study, we used ciglitazone to selectively activate PPARgamma and examine its role in STAR-mediated steroidogenesis in immortalised KK1 mouse granulosa cells and MA-10 mouse Leydig tumour cells. Cotreatment with both dibutyryl-cAMP and ciglitazone revealed a dose-dependent, significant increase in progesterone synthesis, Star promoter activity, Star mRNA and STAR protein relative to either compound alone. The overexpression of PPARgamma further increased Star-promoter activity. The ciglitazone-induced activity of the Star-promoter appears to be mediated through the cAMP-response element half-sites located within its proximal 151 bp. Combined treatment with ciglitazone and dibutyryl-cAMP significantly increased the expression and activity of transcriptional pathways impacted by the activator protein-1 family member c-JUN. The present study demonstrates that ciglitazone and dibutyryl-cAMP synergistically enhance STAR expression in MA-10 and KK1 cells. Ciglitazone-activated PPARgamma appears to increase the sensitivity of Leydig and granulosa cells to cAMP stimulation, possibly via upregulation of c-JUN expression.

Expression of Scavenger Receptor-BI and Low-Density Lipoprotein Receptor and Differential Use of Lipoproteins to Support Early Steroidogenesis in Luteinizing Macaque Granulosa Cells

An ovulatory hCG stimulus to rhesus macaques undergoing controlled ovarian stimulation protocols results in a rapid and sustained increase in progesterone synthesis. The use of lipoproteins as a substrate for progesterone synthesis remains unclear, and the expression of lipoprotein receptors [very-low-density lipoprotein receptor (VLDLR), low-density lipoprotein receptor (LDLR), and scavenger receptor-BI (SR-BI)] soon after human chorionic gonadotropin (hCG) (<12 h) has not been characterized. This study investigated lipoprotein receptor expression and lipoprotein (VLDL, LDL, and HDL) support of steroidogenesis during luteinization of macaque granulosa cells. Granulosa cells were aspirated from rhesus monkeys undergoing controlled ovarian stimulation before or up to 24 h after an ovulatory hCG stimulus. The expression of VLDLR decreased within 3 h of hCG, whereas LDLR and SR-BI increased at 3 and 12 h, respectively. Granulosa cells isolated before hCG were cultured for 24 h in the presence of FSH or FSH plus hCG with or without VLDL, LDL, or HDL. Progesterone levels increased in the presence of hCG regardless of lipoprotein addition, although LDL, but not HDL, further augmented hCG-induced progesterone. Other cells were cultured with FSH or FSH plus hCG without an exogenous source of lipoprotein for 24 h, followed by an additional 24 h culture with or without lipoproteins. Cells treated with hCG in the absence of any lipoprotein were unable to maintain progesterone levels through 48 h, whereas LDL (but not HDL) sustained progesterone synthesis. These data suggest that an ovulatory stimulus rapidly mobilizes stored cholesterol esters for use as a progesterone substrate and that as these are depleted, new cholesterol esters are obtained through an LDLR- and/or SR-BI-mediated mechanism.