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T R Tephly

Publications and source records attributed to T R Tephly.

At least 19 recordsLinked to original sources
PubMed2001

Glucuronidation of linoleic acid diols by human microsomal and recombinant UDP-glucuronosyltransferases: identification of UGT2B7 as the major isoform involved.

Recent reports suggest that linoleic acid (LA) epoxides and diols are associated with important physiological, pharmacological, and pathological events in vivo. We have shown recently that LA-diols are excellent substrates for human liver microsomal UDP-glucuronosyltransferases (UGTs); however, it is not known if other human tissues glucuronidate LA-diols or which UGT isozyme(s) is involved. The present studies with human intestinal microsomes indicate that glucuronidation of LA-diols occurs throughout the gastrointestinal tract, with the highest activity in the small intestine. LA-diols yielded exclusively hydroxyl-linked glucuronides, whereas LA yielded the carboxyl-linked glucuronide. Studies with human recombinant UGTs demonstrated that only UGT2B7 glucuronidated LA and LA-diols. Kinetic analysis with UGT2B7 yielded apparent K(m) values in the range of 40-70 microM and V(max) values from 4.5 to 5.4 nmol/mg x min. These studies indicate that LA and LA-diols are excellent substrates for intestinal UGTs and provide the first evidence for UGT2B7 being the major isoform involved.

Adolescent↗
PubMed2001

Analysis of opioid binding to UDP-glucuronosyltransferase 2B7 fusion proteins using nuclear magnetic resonance spectroscopy.

The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. In this study, we demonstrate that the opioid binding site in UGT2B7 is within the first 119 amino-terminal amino acids. Two maltose binding protein fusion proteins, 2B7F1 and 2B7F2, incorporating the first 157 or 119 amino acids, respectively, of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography. NMR spectroscopy using one-dimensional spectra, the inversion recovery method, and the transferred nuclear Overhauser effect spectroscopy was used to study the binding properties of opioids to the fusion proteins. Morphine was found to bind at a single site within the first 119 amino acids and to undergo a conformational change upon binding, as demonstrated by transferred nuclear Overhauser effect spectroscopy. Dissociation constants were obtained for morphine, naloxone, buprenorphine, and zidovudine, and the results were confirmed by equilibrium dialysis determinations. Two possible opioid binding sites, based on the nearest neighbors from opioid binding to the micro-receptor and to cytochrome 2D6, are proposed.

ATP-Binding Cassette Transporters↗
PubMed2000

4-hydroxyretinoic acid, a novel substrate for human liver microsomal UDP-glucuronosyltransferase(s) and recombinant UGT2B7.

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDP-glucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways.

Glucuronides↗
PubMed2000

UDP-glucuronosyltransferases.

Glucuronidation represents a major pathway which enhances the elimination of many lipophilic xenobiotics and endobiotics to more water-soluble compounds. The UDP-glucuronosyltransferase (UGT) family catalyzes the glucuronidation of the glycosyl group of a nucleotide sugar to an acceptor compound (aglycone) at a nucleophilic functional group of oxygen (eg, hydroxyl or carboxylic acid groups), nitrogen (eg, amines), sulfur (eg, thiols), and carbon, with the formation of a beta-D-glucuronide product. At this time, over 35 different UGT gene products have been described from several different species. UGTs have been divided into two distinct subfamilies based on sequence identities, UGT1 and UGT2. The UGT1 gene subfamily consists of a number of UGTs that result from alternate splicing of multiple first exons and share common exons 2-5. The substrate specificities of the various isoforms have been examined in cultured cell experiments, and include bilirubin, amines, and planar and bulky phenol. The UGT2 gene family is different in that the UGT2 mRNAs are transcribed from individual genes. The UGT2 subfamily consists of numerous enzymes which catalyze the glucuronidation of a diverse chemical base including steroids, bile acids, and opioids. Until recently, the liver has been the major focus for studying the metabolism of xenobiotics and endobiotics. Several groups have identified extrahepatic tissues that express UGT isoforms including the kidney, gastrointestinal tract and brain. This review discusses the two UGT gene families, substrate specificities, and the recent discoveries of UGTs in extrahepatic tissues.

Animals↗
PubMed2000

3'-azido-3'-deoxythimidine (AZT) is glucuronidated by human UDP-glucuronosyltransferase 2B7 (UGT2B7).

3'-Azido-3'-deoxythymidine (AZT) is frequently prescribed to patients infected with the human immunodeficiency virus. After absorption, AZT is rapidly metabolized into 3'-azido-3'-deoxy-5'-glucuronylthymidine by UDP-glucuronosyltransferase (UGT) enzymes. Using labeled [(14)C]UDP-glucuronic acid and microsomal preparations from human kidney 293 cells stably expressing the different human UGT2B isoenzymes, it was demonstrated that AZT glucuronidation is catalyzed specifically by human UGT2B7. The identity of the metabolite formed was confirmed as AZT-G by liquid chromatography coupled with mass spectrometry. UGT2B7 is encoded by a polymorphic gene and kinetic analysis of AZT glucuronidation by the two allelic variants UGT2B7(H(268)) and UGT2B7(Y(268)), yielded apparent K(m) values of 91.0 and 80.1 microM, respectively. Normalization to protein levels yielded glucuronidation efficiency ratios (V(max)/K(m)) of 21.3 and 11.0 microl. min(-1). mg protein(-1) for UGT2B7(H(268)) and UGT2B7(Y(268)), respectively. It remains possible that other UGT enzymes are also involved in AZT conjugation; however, the glucuronidation of AZT by UGT2B7, which is a UGT2B protein expressed in the liver, is consistent with previous findings and supports the physiological relevance of this enzyme in AZT conjugation.

Alleles↗
PubMed2000

Comparative N-glucuronidation kinetics of ketotifen and amitriptyline by expressed human UDP-glucuronosyltransferases and liver microsomes.

Like other basic amphiphilic drugs, the (S)-enantiomer of the antiallergic drug ketotifen exhibited biphasic kinetics when it was converted to two isomeric quaternary ammonium-linked glucuronides in human liver microsomes. For (R)-ketotifen this applied when incubations were carried out in the absence of a detergent. Two UDP-glucuronosyltransferases (UGTs) present in human liver, UGT1A4 and UGT1A3, were previously shown to catalyze tertiary amine N-glucuronidation when expressed in HK293 cells. Therefore, the conjugation kinetics of (R)- and (S)-ketotifen were investigated with the two expressed proteins. When homogenates of HK293 cells expressing UGT1A4 were incubated without detergent, N-glucuronidation kinetics were monophasic with K(M) values of 59 +/- 5 microM for (R)- and 86 +/- 26 microM for (S)-ketotifen. In experiments with membranes containing expressed UGT1A3, somewhat higher K(M) values were obtained. These values correspond to the high rather than to the low K(M) components of ketotifen glucuronidation in liver microsomes, the latter exhibiting K(M) values around 2 and 1 microM, respectively, with (R)- and (S)-ketotifen. With amitriptyline as the substrate, N-glucuronidation kinetics in the absence of detergent were biphasic in human liver microsomes and monophasic with a high K(M) value in cell homogenates containing UGT1A4. The results suggest that UGT1A4 and UGT1A3 catalyze high-K(M) N-glucuronidation of tertiary amine drugs, whereas the low-K(M) reaction requires either an alternative enzyme or a special conformation of UGT1A4 or UGT1A3 that can be attained in liver microsomes, but not in HK293 cell membranes.

Amitriptyline↗
PubMed1999

Expression of UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain and identification of 5-hydroxytryptamine as a substrate.

The extrahepatic expression of UDP-glucuronosyltransferases (UGTs) is important in the detoxification of a number of endogenous and exogenous compounds, including 5-hydroxytryptamine and morphine. Studies were designed to investigate the extrahepatic expression of human UGTs using RT-PCR techniques and to determine the UGTs involved in the glucuronidation of 5-hydroxytryptamine. Human UGT2B7 expression was found in the human liver, kidney, pancreas, and brain, while UGT1A6 expression is found in the liver, kidney, and brain. This is the first observation of UGTs present in the human central nervous system. Using glucuronidation assays, a significant amount of 5-hydroxytryptamine glucuronide was found to be catalyzed by UGT1A6. These studies suggest that UGT2B7 may play an important role in the overall contribution of morphine analgesia by serving to generate the potent morphine-6-O-glucuronide in situ. UGT1A6 could play an important role in the glucuronidation of 5-hydroxytryptamine in vivo, therefore terminating the actions of the neurotransmitter.

Adult↗
PubMed1999

Comments on the purported generation of formaldehyde and adduct formation from the sweetener aspartame.

A recent paper by Trocho et al. (1) describes experiments meant to show that formaldehyde adducts are formed when rats are administered the sweetener aspartame. These authors assume that the methanol carbon of aspartame generates formaldehyde which then forms adducts with protein, DNA, and RNA. Doses employed range widely. In this letter, studies which have been published previously and which were not cited by these authors are reviewed in order to put into perspective the disposition of methanol and formaldehyde in monkeys and humans, species relevant to the toxicity of methanol and its toxic metabolite, formic acid.

Animals↗
PubMed1999

Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

Animals↗
PubMed1999

Symposium overview: Characterization of xenobiotic metabolizing enzyme function using heterologous expression systems.

Genetically modified cell lines can be very useful models for assessing the toxicologic effects of modulation of expression of individual gene products in comparison to their isogenic parental control cell lines. This symposium begins with an overview of general issues related to development and utilization of model systems created by transfection of cell lines to induce elevated expression of metabolic enzymes of toxicologic relevance. Selected studies that illustrate the heterologous expression rationale and various approaches to transgenic-cell model construction are represented. Results to date with cells engineered to express specific transfected genes are discussed, with emphasis on the effects of expression of selected phase I or phase II enzymes on cellular sensitivity to several toxic end-points. The individual sections highlight the utility of these model cell lines for examining the role of enzyme catalysis and function in metabolism of biologically active xenobiotic or endobiotic compounds of interest in toxicology. Both activating and detoxifying enzymes are discussed, with principal emphasis on the latter. This symposium includes talks on transfected cells that express aldehyde dehydrogenases, superoxide dismutase, UDP-glycosyltransferases, glutathione transferases, and cytochrome P450 isozymes. In addition to the general toxicologic utility and advantages of these genetically engineered cell lines, this overview emphasizes their particular contributions to the insights obtained to date with the specific model cell lines.

Animals↗
PubMed1999

Folate and folate-dependent enzymes associated with rat CNS development.

Folic acid and its derivatives are important mediators in growth-related cellular processes. The concentration of folate and two folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (10-FTHFS) and 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH), was determined in brain regions over the early period of rat development. Folate concentrations determined at birth were high in all brain regions studied. During the first 2 weeks, folate concentrations declined steadily, followed by a period of significant increase. High and invariant activity of 10-FTHFS was found throughout the period of study. Low amounts of 10-FTHFDH were seen for the first 2 weeks, but increased significantly from postnatal days 14 to 28. These changes correlated with changes determined in the concentration of folate, supporting the idea that this protein is involved with folate uptake and/or storage. Furthermore, immunohistochemical expression of 10-FTHFDH in different rat brain regions revealed glial cells as a preferential cellular location for this folate-binding protein.

Aging↗
PubMed1999

Glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by rat UDP-glucuronosyltransferase 2B1.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of NNAL is a potentially important detoxification pathway for these carcinogens. To identify the UGT isozyme(s) involved in this pathway, we examined the glucuronidation of NNAL in rat liver microsomes and homogenates from cell lines overexpressing specific UGT isozymes. NNAL glucuronidation was induced in liver microsomes from rats treated with family 2 UGT inducers including phenobarbitol and 3, 5-di-tert-butyl-4-hydroxytoluene, which exhibited 1.7- and 2.6-fold higher rates of glucuronidation than microsomes from control rats. The rates of NNAL glucuronidation in liver microsomes from GUNN (deficient in family 1 UGTs) and RHA parental control rats were similar. All rat liver microsomes used in the present study catalyzed the glucuronidation of (S)-NNAL at a rate between 3.5 and 5.5 times that of the glucuronidation of (R)-NNAL. Liver microsomes from Wistar rats exhibiting the low-androsterone glucuronidation phenotype characteristic of the UGT2B2-deficient genotype glucuronidated NNAL at a rate similar to microsomes from Wistar rats exhibiting the high-androsterone glucuronidation phenotype/UGT2B2 [+] genotype. Homogenates from UGT2B1-overexpressing cells catalyzed the glucuronidation of NNAL at a K(m) of 745 microM. As with rat liver microsomes, NNAL-Gluc I was the major diastereomer formed by UGT2B1. Glucuronidation of NNAL was not detected with homogenates from UGT2B12-overexpressing cells. These results suggest that UGT2B1 plays an important role in the glucuronidation of NNAL in the rat.

Animals↗
PubMed1999

Studies on the substrate specificity of human intestinal UDP- lucuronosyltransferases 1A8 and 1A10.

Although the liver has been considered the most important organ involved in glucuronidation, recent studies have focused on the role of the gastrointestinal tract in the glucuronidation of xenobiotics and endobiotics. Two UDP-glucuronosyltransferase (UGT) isoforms of human intestinal mucosa, which are absent in liver, have been identified by reverse transcriptase-polymerase chain reaction. mRNAs of UGT1A8 and UGT1A10 were detected in both the small intestine and the colon. The corresponding cDNAs for UGT1A8 and UGT1A10 were cloned from ileal RNA and inserted into the mammalian expression vector pcDNA3. Transfection of the cDNAs into human embryonic kidney 293 cells was carried out and stable expression was achieved. Membrane preparations from human embryonic kidney 293 cells expressing either UGT1A8 or UGT1A10 were isolated and the expression of each isoform was analyzed by Western blot. The catalytic activity of stably expressed UGT1A8 toward catechol estrogens, coumarins, flavonoids, anthraquinones, and phenolic compounds was much higher than that of UGT1A10. UGT1A8, but not UGT1A10, catalyzed the glucuronidation of opioids, bile acids, fatty acids, retinoids, and clinically useful drugs, such as ciprofibrate, furosemide, and diflunisal. These studies suggest that human intestinal UGTs may play an important role in the detoxification of xenobiotic compounds and, in some cases, limit the bioavailability of therapeutic agents.

Biological Availability↗
PubMed1998

UDP-glucuronosyltransferases in human intestinal mucosa.

While UDP-glucuronosyltransferases (UGTs) are known to be expressed at high levels in human liver, relatively little is known about extrahepatic expression. In the present study, UGT2B family isoforms involved in the glucuronidation of steroid hormones and bile acids have been characterized in microsomes prepared from jejunum, ileum and colon from six human subjects. Glucuronidation of androsterone and testosterone was highly significant and increased from proximal to distal intestine. In contrast, hyodeoxycholic acid was glucuronidated at a low level in jejunum and ileum and activity was barely detectable in colon. No significant glucuronidation of lithocholic acid was found. Small phenols were glucuronidated with much lower activity than found in liver. High levels of UGT protein were detected with polyclonal anti-rat androsterone- and testosterone-UGT antibodies, whereas UGT2B4, a major hepatic hyodeoxycholic acid-specific UGT, was undetectable using a highly specific anti-human UGT2B4 antibody. Screening for RNA expression by RT-PCR confirmed the absence of UGT2B4 and UGT1A6 and showed expression of UGT2B7, a hepatic isoform shown to glucuronidate androsterone, in all intestinal segments. To our knowledge, the presence of functional androsterone and testosterone directed isoforms in human intestine is a novel finding which supports the idea that the intestinal tract functions as a steroid-metabolizing organ and plays a significant role in steroid hormone biotransformation.

Adult↗
PubMed1998

Cloning and expression of human UDP-glucuronosyltransferase (UGT) 1A8.

The mRNA expression of human UDP-glucuronosyltransferase 1A8 (UGT1A8) has been found in jejunum, ileum, and colon but not in liver. A cDNA with a complete UGT1A8 coding region was amplified from total human ileal RNA by reverse transcriptase-polymerase chain reaction and inserted into the mammalian expression vector, pcDNA3. Lysates of HK293 cells expressing UGT1A8 revealed the expression of a protein with a molecular mass of 56 kDa by Western blot analysis. Transiently expressed human UGT1A8 shows glucuronidation activities with coumarins, anthraquinones, flavonoids, phenolic compounds, catechol estrogens, 17-hydroxyandrogens, primary amines such as the carcinogen 4-aminobiphenyl, and certain opioids. This UGT may play an important role in the detoxification of xenobiotics in human intestine.

Catalysis↗
PubMed1998

Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes.

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.

Animals↗
PubMed1998

Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7.

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Estradiol↗