Monocots and eudicots have more conservative flower water use strategies than basal angiosperms.

Water balance is crucial for the growth and flowering of plants. However, the mechanisms by which flowers maintain water balance are poorly understood across different angiosperm branches. Here, we investigated 29 floral hydraulic and economic traits in 24 species from ANA grade, magnoliids, monocots, and eudicots. Our main objective was to compare differences in flower water use strategies between basal angiosperms (ANA grade and magnoliids) and derived group (monocots and eudicots). We found that basal angiosperms had richer petal stomatal density, higher pedicel hydraulic diameter, and flower mass per area, but lower pedicel vessel wall reinforcement and epidermal cell thickness compared to monocots and eudicots. We also observed significant trade-offs and coordination among different floral traits. Floral traits associated with reproduction, such as floral longevity and size, were strongly linked with physiological and anatomical traits. Our results systematically reveal the variation in flower economic and hydraulic traits from different angiosperm branches, deepening understanding of flower water use strategies among these plant taxa. We conclude that basal angiosperms maintain water balance with high water supply, whereas monocots and eudicots maintain a more conservative water balance.

A bibliometric analysis for relapsed/refractory Non-Hodgkin lymphoma.

OBJECTIVE: This study aimed to analyze the current research status and trends of publications on relapsed/refractory Non-Hodgkin lymphoma (r/r NHL) using CiteSpace software and to know which centers and authors we should follow in the first place while doing research on r/r NHL. MATERIALS AND METHODS: The publications were retrieved from the Web of Science Core collection database, and CiteSpace (5.5.R5) software was used to analyze the authors, institutions, countries, and keywords. RESULTS: A total of 567 publications from 2009 to 2021 were retrieved, and the most fertile authors, institutions, nationalities and keywords in the field of r/r NHL were identified. Pier Luigi Zinzani team, Kensei Tobinai team, Andre Goy team, and Julie M. Vose team are recognized the main research teams in this field. USA makes the greatest contribution having research funds for r/r NHL. Key cluster areas of research include mantle cell lymphoma, pathway, lymphoma, relapse, pixantrone, Non-Hodgkin lymphoma, romidepsin, relapsed, T-cell lymphoma, and activated T cells. According to the keywords' timeline, the research trends of r/r NHL changed from bone marrow transplantation, radioimmunotherapy, chemotherapy to novel target drugs (like ibritumomab tiuxetan, inhibitor) and criteria EBM. CONCLUSIONS: The bibliometric study provides insights into hotspots and trends in the field of r/r NHL in the past 12 years. It serves us to extract useful information from complex data and provide information for clinicians and researchers.

The efficacy and safety of canagliflozin in the treatment of patients with early diabetic nephropathy.

This study aimed to observe the efficacy and safety of canagliflozin in the treatment of patients with early diabetic nephropathy (DN) and investigate its effect in reducing urinary protein levels. A total of 132 patients with DN and normal renal function (estimated glomerular filtration rate >60 ml/min/1.73 m2) combined with urine albumin/creatinine ratio (UACR) no less than 30 mg/g were selected and randomly divided into a control group and an observation group, with 66 cases in each group. Irbesartan treatment was administered to the control group based on conventional treatment, while a combination of canagliflozin and irbesartan was given to the observation group based on conventional treatment. The changes in blood glucose, blood pressure, body weight, renal function, and urinary protein were observed in both groups. Compared with the control group, patients in the observation group showed a significant decrease in blood glucose, blood pressure, body weight, and urinary protein starting at week 4 of treatment and continuing until the end of the experiment at week 24 (all P<0.05). Within the observation group, blood glucose, blood pressure, body weight, and urinary protein decreased significantly with 24 weeks of treatment compared with those before the experiment (P<0.01). Patients in the observation group experienced a mild decrease in renal function at week 4, but the function began to gradually recover by week 8 and had returned to the baseline by the end of the study (P<0.05). In conclusion: canagliflozin has good efficacy and safety in the treatment of early DN. It also lowers urinary protein levels and blood glucose.

Direct acceleration of collimated monoenergetic sub-femtosecond electron bunches driven by a radially polarized laser pulse.

High-quality ultrashort electron beams have diverse applications in a variety of areas, such as 4D electron diffraction and microscopy, relativistic electron mirrors and ultrashort radiation sources. Direct laser acceleration (DLA) mechanism can produce electron beams with a large amount of charge (several to hundreds of nC), but the generated electron beams usually have large divergence and wide energy spread. Here, we propose a novel DLA scheme to generate high-quality ultrashort electron beams by irradiating a radially polarized laser pulse on a nanofiber. Since electrons are continuously squeezed transversely by the inward radial electric field force, the divergence angle gradually decreases as electrons transport stably with the laser pulse. The well-collimated electron bunches are effectively accelerated by the circularly-symmetric longitudinal electric field and the relative energy spread also gradually decreases. It is demonstrated by three-dimensional (3D) simulations that collimated monoenergetic electron bunches with 0.75° center divergence angle and 14% energy spread can be generated. An analytical model of electron acceleration is presented which interprets well by the 3D simulation results.

The lack of HSD17B3 in male mice results in disturbed Leydig cell maturation and endocrine imbalance akin to humans with HSD17B3 deficiency.

Hydroxysteroid (17ß) dehydrogenase type 3 (HSD17B3) deficiency causes a disorder of sex development in humans, where affected males are born with female-appearing external genitalia, but are virilized during puberty. The hormonal disturbances observed in the Hsd17b3 knockout mice (HSD17B3KO), generated in the present study, mimic those found in patients with HSD17B3 mutations. Identical to affected humans, serum T in the adult HSD17B3KO mice was within the normal range, while a striking increase was detected in serum A-dione concentration. This resulted in a marked reduction of the serum T/A-dione ratio, a diagnostic hallmark for the patients with HSD17B3 deficiency. However, unlike humans, male HSD17B3KO mice were born with normally virilized phenotype, but presenting with delayed puberty. In contrast to the current belief, data from HSD17B3KO mice show that the circulating T largely originates from the testes, indicating a strong compensatory mechanism in the absence of HSD17B3. The lack of testicular malignancies in HSD17B3KO mice supports the view that testis tumors in human patients are due to associated cryptorchidism. The HSD17B3KO mice presented also with impaired Leydig cell maturation and signs of undermasculinization in adulthood. The identical hormonal disturbances between HSD17B3 deficient knockout mice and human patients make the current mouse model valuable for understanding the mechanism of the patient phenotypes, as well as endocrinopathies and compensatory steroidogenic mechanisms in HSD17B3 deficiency.

Melatonin regulates mitochondrial function and biogenesis during rat dental papilla cell differentiation.

OBJECTIVE: The aim of this study was to investigate the effect of melatonin on mitochondria of dental papilla cells (DPCs) during the odontogenic differentiation process. MATERIALS AND METHODS: Primary DPCs were obtained from the first molar dental papilla of neonatal rats and cultured in osteogenic (OS) or basal medium supplemented with melatonin at different concentrations (0, 1 pM, 0.1 nM, 10 nM, and 1 µM) for differentiation in vitro. Effects of melatonin on differentiation, mitochondrial respiratory function, and mitochondrial biogenesis of DPCs were analyzed. RESULTS: Upon odontogenic induction, Alkaline phosphatase (ALP) activity, dentin sialophosphoprotein (DSPP), and dentin matrix protein (DMP1) expression were significantly enhanced, with a peaked expression at 10 nM of melatonin treatment. During DPCs differentiation, 10 nM melatonin could significantly induce the increase of intracellular Adenosine triphosphate (ATP), the decrease of the oxidized form of nicotinamide adenine dinucleotide (NAD+)/NADH ratio and reactive oxygen species (ROS). The mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM) were significantly increased, and the peak level of expression was found in cells treated with 10 nM of melatonin. Furthermore, the mitochondria DNA (mtDNA) copy number was significantly decreased during DPCs differentiation. CONCLUSIONS: These findings suggest that melatonin can promote the differentiation of rat DPCs and regulate mitochondrial energy metabolism, ROS scavenging, and mitochondrial biogenesis.

[The effect of air test and methylene blue perfusion test on detecting the quality of anastomosis during laparoscopic rectal cancer excision (Dixon)].

Objective: To investigate the feasibility and safety of air test (AT) and methylene blue perfusion test (MBPT) to detect the quality of the anastomosis in laparoscopic rectal cancer excision (Dixon), and compare the two approaches. Methods: AT is performed by filling the pelvis with saline solution and insufflating the rectum with air through a size 22 G balloon catheter (Foley). MBPT is carried out by surrounding clean sponges around anastomosis and injecting methylene blue solution into the rectum as like as AT. The balloon catheter connected manometer,ensuring the pressure in rectum can reach 40 cmH(2)O during AT and MBPT. The presence of air bubbles and overt blue-stained spillage indicated anastomotic leaks which are were resolved during surgery. All 28 patients undergoing laparoscopic rectal excision received both AT and MBPT intraoperatively in a randomized fashion. The integrity of the anastomosis, postoperative vital signs, blood examination, drainage and postoperative imaging were analyzed. Results: All 28 patients received both tests successfully with no adverse event. MBPT Level 1 was detected in 15 cases, level 2 in 8 cases, level 3 in 5 cases. No MBPT level 4 was observed. AT level 1 was detected in 22 cases, level 2 in 5 cases, level 3 in 1 cases. No AT level 4 was founded. Three cases were diagnosed with postoperative anastomotic leakage (3/28, 10.71%), of which 2 cases were Grade B [definition and grading proposed by the international study group of rectal cancer (ISREC) in 2010]. One case was Grade C. The positive rate of MBPT was superior to AT (the McNemar testing, P<0.01). Conclusions: The two intraoperative tests are both technically feasible and safe. Compared to AT, MBPT has the advantage of localizing the leak site with a higher positive accuracy, and represents a promising standardized approach for intraoperative test of the anastomosis quality. Intraoperative repair is absolutely helpful for the level 3 and 4 intraoperative tests.

Defects in chondrocyte maturation and secondary ossification in mouse knee joint epiphyses due to Snorc deficiency.

OBJECTIVE: The role of Snorc, a novel cartilage specific transmembrane proteoglycan, was studied during skeletal development using two Snorc knockout mouse models. Hypothesizing that Snorc, like the other transmembrane proteoglycans, may be a coreceptor, we also studied its interaction with growth factors. METHODS: Skeletal development was studied in wild type (WT) and Snorc knockout mice during postnatal development by whole mount staining, X-ray imaging, histomorphometry, immunohistochemistry and qRT-PCR. Snorc promoter activity was studied by applying the LacZ reporter expressed by the targeting construct. Slot blot binding and cell proliferation assays were used to study the interaction of Snorc with several growth factors. RESULTS: Snorc expression was localized in the knee epiphyses especially to the prehypertrophic chondrocytes delineating the cartilage canals and secondary ossification center (SOC). Snorc was demonstrated to have a glycosaminoglycan independent affinity to FGF2 and it inhibited FGF2 dependent cell growth of C3H101/2 cells. In Snorc deficient mice, SOCs in knee epiphyses were smaller, and growth plate (GP) maturation was disturbed, but total bone length was not affected. Central proliferative and hypertrophic zones were enlarged with higher extracellular matrix (ECM) volume and rounded chondrocyte morphology at postnatal days P10 and P22. Increased levels of Ihh and Col10a1, and reduced Mmp13 mRNA expression were observed at P10. CONCLUSIONS: These findings suggest a role of Snorc in regulation of chondrocyte maturation and postnatal endochondral ossification. The interaction identified between recombinant Snorc core protein and FGF2 suggest functions related to FGF signaling.

Generation of membrane-bound catechol-O-methyl transferase deficient mice with disctinct sex dependent behavioral phenotype.

Catechol-O-methyltransferase (COMT) has two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-MT), anchored to intracellular membranes. COMT is involved in the O-methylation of L-DOPA, dopamine and other catechols. The exact role of MB-COMT is still mostly unclear. We wanted to create a novel genetically modified mouse model that specifically lacks MB-COMT activity and to study their behavioral phenotype. MB-COMT knock-in mutant mice were generated by introducing two point mutations in exon 2 of the Comt gene (ATGCTG->GAGCTC disabling the function of the P2 promoter and allowing only the P1-regulated S-COMT transcription. The first mutation changes methionine to glutamic acid whereas the second one does not affect coding. The expression of the two COMT isoforms, total COMT activity in several areas of the brain and peripheral tissues and extracellular dopamine concentrations after L-DOPA (10 mg/kg) and carbidopa (30 mg/kg) subcutaneous administration were assessed. A battery of behavioral tests was performed to compare MB-COMT deficient mice and their wild type littermates of both sexes. MB-COMT deficient mice were seemingly normal, bred usually and had unaltered COMT activity in the brain and periphery despite a complete lack of the MB-COMT protein. MB-COMT deficient male mice showed higher extracellular dopamine levels than their wild-type littermates in the striatum, but not in the mPFC. In addition, the MB-COMT deficient male mice exhibited a distinct endophenotype characterized by schizophrenia-related behaviors like aggressive behavior and reduced prepulse inhibition. They also had prolonged immobility in the tail suspension test. Both sexes were sensitized to acute pain and had normal motor activity but disturbed short-term memory. Hence the behavioral phenotype was not limited to schizophrenia-related endophenotype and some behavioural findings were not sex-dependent. Our findings indicate that MB-COMT is critical for behavior, and its function in COMT-dependent brain areas cannot be entirely substituted by the remaining S-COMT.

Scanning electron microscopy studies of the antennal sensilla of Metaphycus parasaissetiae Zhang & Huang (Hymenoptera: Encyrtidae).

Metaphycus parasaissetiae Zhang & Huang (Hymenoptera: Encyrtidae) is an important adult parasitoid of Parasaissetia nigra Nietner (Hemiptera: Coccoidea). The external morphology of the antennal sensilla of male and female M. parasaissetiae was examined using scanning electron microscopy. The geniculate antennae of male and female M. parasaissetiae were composed of a scape with a basal radicula, a barrel-shaped pedicel, and a long flagellum. Twelve morphologically distinct types of sensilla were identified, including multiporous placoid sensilla, campaniform sensilla, finger-like sensilla, multiporous basiconic sensilla (BS-1), three aporous types of basiconic sensilla (BS-2, BS-3, and BS-4), two types of aporous trichoid sensilla (TS-1 and TS-3), a type of multiporous trichoid sensilla (TS-2), and two types of sensilla chaetica (CH-1 and CH-2). Sex dimorphism in the sensilla composition of M. parasaissetiae is also observed. Major differences between the sexes were found in the number, distribution, shape, structure, and size of the identified sensilla. We also discuss on the functional aspects of these sensilla to elucidate the mechanisms involved in host searching and courtship behavior of M. parasaissetiae.

Study on mating ecology and sex ratio of three internally ovipositing fig wasps of Ficus curtipes.

Studies on mating ecology and sex allocation in fig-parasitizing wasps ovipositing from outside the fig have given valuable insights into known factors that are responsible for the theory of sex ratio. Similarly, internally ovipositing fig-parasitizing wasps and fig-pollinating wasps provide interesting models for comparative analysis. In addition to the fig-pollinating wasp Eupristina sp., we found that Ficus curtipes hosts two species of internally ovipositing fig-parasitizing wasps: D. yangi and Lipothymus sp. Eupristina sp. males showed less aggression. Eupristina sp. has wingless males that mate only within the natal patch, providing excellent examples of full local-mate competition. D. yangi males showed high levels of aggression and lethal combat. D. yangi has winged males but mate mostly within the natal patch. Only a few matings occur after male dispersal. Its sex ratio was lower than the prediction of partial local mate competition theory. Wingless male Lipothymus sp., which mate partly after dispersal, did not present fatal fight. Therefore, the mating behaviour of D. yangi and Lipothymus sp. did not follow predicted patterns, based on wing morph. The mating pattern of D. yangi and Lipothymus sp. should follow the partial local mate competition theory. Furthermore, there was not a significant correlation between the proportion of males and the proportion of fruit parasitized in both winged D. yangi males and wingless Lipothymus sp. males.

Numerical simulation of municipal solid waste combustion in a novel two-stage reciprocating incinerator.

A mathematical model was presented in this paper for the combustion of municipal solid waste in a novel two-stage reciprocating grate furnace. Numerical simulations were performed to predict the temperature, the flow and the species distributions in the furnace, with practical operational conditions taken into account. The calculated results agree well with the test data, and the burning behavior of municipal solid waste in the novel two-stage reciprocating incinerator can be demonstrated well. The thickness of waste bed, the initial moisture content, the excessive air coefficient and the secondary air are the major factors that influence the combustion process. If the initial moisture content of waste is high, both the heat value of waste and the temperature inside incinerator are low, and less oxygen is necessary for combustion. The air supply rate and the primary air distribution along the grate should be adjusted according to the initial moisture content of the waste. A reasonable bed thickness and an adequate excessive air coefficient can keep a higher temperature, promote the burnout of combustibles, and consequently reduce the emission of dioxin pollutants. When the total air supply is constant, reducing primary air and introducing secondary air properly can enhance turbulence and mixing, prolong the residence time of flue gas, and promote the complete combustion of combustibles. This study provides an important reference for optimizing the design and operation of municipal solid wastes furnace.

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

Novel expression and functional role of ghrelin in rat testis.

Ghrelin, the endogenous ligand for the GH-secretagogue receptor (GHS-R), is a recently cloned peptide, primarily expressed in the stomach and hypothalamus, that acts at central levels to elicit GH release and, notably, to regulate food intake. However, the possibility of additional, as yet unknown, peripheral effects of ghrelin cannot be ruled out. In the present communication, we provide evidence for the novel expression of ghrelin and its functional receptor in rat testis. Testicular ghrelin gene expression was demonstrated throughout postnatal development, and ghrelin protein was detected in Leydig cells from adult testis specimens. Accordingly, ghrelin mRNA signal became undetectable in rat testis following selective Leydig cell elimination. In addition, testicular expression of the gene encoding the cognate ghrelin receptor was observed from the infantile period to adulthood, with the GHS-R mRNA being persistently expressed after selective withdrawal of mature Leydig cells. From a functional standpoint, ghrelin, in a dose-dependent manner, induced an average 30% inhibition of human CG- and cAMP-stimulated T secretion in vitro. This inhibitory effect was associated with significant decreases in human CG-stimulated expression levels of the mRNAs encoding steroid acute regulatory protein, and P450 cholesterol side-chain cleavage, 3beta-hydroxy steroid dehydrogenase, and 17beta-hydroxy steroid dehydrogenase type III enzymes. Overall, our data are the first to provide evidence for a possible direct action of ghrelin in the control of testicular function. Furthermore, the present results underscore an unexpected role of ghrelin as signal with ability to potentially modulate not only growth and body weight homeostasis but also reproductive function, a phenomenon also demonstrated recently for the adipocyte-derived hormone, leptin.

Analytical methodologies for aluminium speciation in environmental and biological samples--a review.

It is recognized that aluminium (Al) is a potential environmental hazard. Acidic deposition has been linked to increased Al concentrations in natural waters. Elevated levels of Al might have serious consequences for biological communities. Of particular interest is the speciation of Al in aquatic environments, because Al toxicity depends on its forms and concentrations. In this paper, advances in analytical methodologies for Al speciation in environmental and biological samples during the past five years are reviewed. Concerns about the specific problems of Al speciation and highlights of some important methods are elucidated in sections devoted to hybrid techniques (HPLC or FPLC coupled with ET-AAS, ICP-AES, or ICP-MS), flow-injection analysis (FIA), nuclear magnetic resonance (27Al NMR), electrochemical analysis, and computer simulation. More than 130 references are cited.

Molecular mechanisms of leptin action in adult rat testis: potential targets for leptin-induced inhibition of steroidogenesis and pattern of leptin receptor messenger ribonucleic acid expression.

Leptin, the product of the ob gene, is a pivotal signal in the regulation of neuroendocrine function and fertility. Although much of the action of leptin in the control of the reproductive axis is exerted at the hypothalamic level, some direct effects of leptin on male and female gonads have also been reported. Indeed, recent evidence demonstrated that leptin is able to inhibit testosterone secretion at the testicular level. However, the molecular mechanisms behind this effect remain unclear. The focus of this study was twofold: (1) to identify potential targets for leptin-induced inhibition of steroidogenesis, and (2) to characterize in detail the pattern of expression and cellular distribution of leptin receptor (Ob-R) mRNA in adult rat testis. In pursuit of the first goal, slices of testicular tissue from adult rats were incubated with increasing concentrations of recombinant leptin (10(-9)--10(-7 )M) in the presence of human chorionic gonadotropin (hCG; 10 IU/ml). In this setting, testosterone secretion in vitro was monitored, and expression levels of mRNAs encoding steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450 scc) and 17 beta-hydroxysteroid dehydrogenase type III (17 beta-HSD) were assessed by Northern hybridization. In pursuit of the second goal, the pattern of cellular expression of the Ob-R gene in adult rat testis was evaluated by in situ hybridization using a riboprobe complementary to all Ob-R isoforms. In addition, testicular expression levels of the different Ob-R isoforms, previously identified in the hypothalamus, were analyzed by means of semi-quantitative RT-PCR. In keeping with our previous data, recombinant leptin significantly inhibited hCG-stimulated testosterone secretion. In this context, leptin, in a dose-dependent manner, was able to co-ordinately decrease the hCG-stimulated expression levels of SF-1, StAR and P450 scc mRNAs, but it did not affect those of 17 beta-HSD type III. In situ hybridization analysis showed a scattered pattern of cellular expression of the Ob-R gene within the adult rat testis, including Leydig and Sertoli cells. In addition, assessment of the pattern of expression of Ob-R subtypes revealed that the long Ob-Rb isoform was abundantly expressed in adult rat testis. However, variable levels of expression of Ob-Ra, Ob-Re, and Ob-Rf mRNAs were also detected, whereas those of the Ob-Rc variant were nearly negligible. In conclusion, our results indicate that decreased expression of mRNAs encoding several up-stream elements in the steroidogenic pathway may contribute, at least partially, to leptin-induced inhibition of testicular steroidogenesis. In addition, our data on the pattern of testicular expression of Ob-R isoforms and cellular distribution of Ob-R mRNA may help to further elucidate the molecular mechanisms of leptin action in rat testis.

Developmental and hormonal regulation of leptin receptor (Ob-R) messenger ribonucleic acid expression in rat testis.

In target tissues, leptin receptor (Ob-R) gene expression results in an array of alternatively spliced isoforms (Ob-Ra to Ob-Rf) with different functional features. Recent evidence has pointed to a direct role of leptin in the control of testicular function. However, complete elucidation of the pattern of Ob-R gene expression in the male gonad is still pending. The focus of this study was to characterize in detail the developmental pattern of expression and hormonal regulation of Ob-R gene in rat testis. To this end, the overall expression of Ob-R mRNA was compared to that of the fully functional, long Ob-Rb isoform in different experimental settings, using semiquantitative reverse transcription-polymerase chain reaction. Expression of Ob-R mRNA was detected in testes from 15-, 30-, 45-, and 75-day-old rats at rather constant relative levels. In contrast, testicular expression of Ob-Rb mRNA was higher in pubertal testes (15- to 30-day-old rats) and declined in adulthood. In testes from 30-day-old animals, analysis of isoform distribution revealed that, in addition to abundant Ob-Rb mRNA levels, expression of Ob-Ra, Ob-Rf, and, to a lesser extent, Ob-Rc and Ob-Re messages is detected. Testicular Ob-R mRNA expression appeared sensitive to neonatal imprinting as neonatal treatment with estradiol benzoate (500 microg/rat; Day 1 postpartum) resulted in a persistent increase (P: < 0.01) in the relative expression level of Ob-R mRNA, a phenomenon only partially mimicked by neonatal suppression of serum gonadotropins by means of LHRH-antagonist administration. In addition, neonatal estrogenization differentially altered the pattern of expression of Ob-R isoforms in adult rat testis, as expression of Ob-Rb mRNA was decreased to undetectable levels, whereas that of Ob-Rc remained unaltered, and Ob-Ra, Ob-Rf, and, to a lesser extent, Ob-Re mRNA levels were significantly increased (P: < 0.01) by neonatal exposure to estrogen. Finally, down-regulation of testicular Ob-R gene expression by homologous and heterologous signals was demonstrated as relative levels of Ob-R and Ob-Rb mRNAs were significantly decreased (P: < 0.01), in a coordinate manner, in rat testis after exposure to human recombinant leptin in vitro, and after stimulation with hCG and FSH in vivo. In conclusion, our results indicate that testicular Ob-R gene expression is developmentally regulated, imprinted by the neonatal endocrine milieu, and sensitive to regulation by leptin and gonadotropins. The ability of pivotal signals in testicular function to regulate Ob-R gene expression further supports the contention of a direct role of leptin in functional control of the rat testis.

Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice.

To study further the role of gonadotropins in reproductive functions, we generated mice with LH receptor (LHR) knockout (LuRKO) by inactivating, through homologous recombination, exon 11 on the LHR gene. LuRKO males and females were born phenotypically normal, with testes, ovaries, and genital structures indistinguishable from their wild-type (WT) littermates. Postnatally, testicular growth and descent, and external genital and accessory sex organ maturation, were blocked in LuRKO males, and their spermatogenesis was arrested at the round spermatid stage. The number and size of Leydig cells were dramatically reduced. LuRKO females also displayed underdeveloped external genitalia and uteri postnatally, and their age of vaginal opening was delayed by 5-7 days. The (-/-) ovaries were smaller, and histological analysis revealed follicles up to the early antral stage, but no preovulatory follicles or corpora lutea. Reduced gonadal sex hormone production was found in each sex, as was also reflected by the suppressed accessory sex organ weights and elevated gonadotropin levels. Completion of meiosis of testicular germ cells in the LuRKO males differs from other hypogonadotropic/cryptorchid mouse models, suggesting a role for FSH in this process. In females, FSH appears to stimulate developing follicles from the preantral to early antral stage, and LH is the stimulus beyond this stage. Hence, in each sex, the intrauterine sex differentiation is independent of LH action, but it has a crucial role postnatally for attaining sexual maturity. The LuRKO mouse is a close phenocopy of recently characterized human patients with inactivating LHR mutations, although the lack of pseudohermaphroditism in LuRKO males suggests that the intrauterine sex differentiation in this species is not dependent on LH action.

Elevated luteinizing hormone induces expression of its receptor and promotes steroidogenesis in the adrenal cortex.

Transgenic (TG) female mice expressing bLHbeta-CTP (a chimeric protein derived from the beta-subunit of bovine luteinizing hormone [LH] and a fragment of the beta-subunit of human chorionic gonadotropin [hCG]) exhibit elevated serum LH, infertility, polycystic ovaries, and ovarian tumors. In humans, increased LH secretion also occurs in infertility and polycystic ovarian syndrome, often concomitant with adrenocortical dysfunction. We therefore investigated adrenal function in LH overexpressing bLHbeta-CTP female mice. The size of their adrenals was increased by 80% with histological signs of cortical stimulation. Furthermore, adrenal steroid production was increased, with up to 14-fold elevated serum corticosterone. Primary adrenal cells from TG and control females responded similarly to ACTH stimulation, but, surprisingly, the TG adrenals responded to hCG with significantly increased cAMP, progesterone, and corticosterone production. LH receptor (LHR) expression and activity were also elevated in adrenals from female TG mice, but gonadectomized TG females showed no increase in corticosterone, suggesting that the dysfunctional ovaries of the intact TG females promote adrenocortical hyperfunction. We suggest that, in intact TG females, enhanced ovarian estrogen synthesis causes increased secretion of prolactin (PRL), which elevates LHR expression. Chronically elevated serum LH, augmented by enhanced PRL production, induces functional LHR expression in mouse adrenal cortex, leading to elevated, LH-dependent, corticosterone production. Thus, besides polycystic ovaries, the bLHbeta-CTP mice provide a useful model for studying human disorders related to elevated LH secretion and adrenocortical hyperfunction.

A common polymorphic allele of the human luteinizing hormone beta-subunit gene: additional mutations and differential function of the promoter sequence.

A common genetic variant (V) of the human luteinizing hormone (LH) beta-subunit gene was recently discovered. The V-LH molecules have higher bioactivity in vitro, but shorter half-life in circulation, which apparently is related to the alterations of LH function observed in individuals homo- and heterozygous for the V-LHbeta allele. We have now studied whether additional mutations in the V-LHbeta promoter sequence could contribute to the altered physiology of the LH variant molecules. The 661 bp 5'-flanking region of the V-LHbeta gene, retrieved from human genomic DNA by PCR, contained eight single-nucleotide changes, as compared with the wild-type (wt) LHbeta promoter. The finding was consistent in DNA samples of different ethnic groups. Reporter constructs with various lengths of the wt- and V-LH promoter sequences, driving the firefly luciferase reporter gene, were transfected into an immortalized mouse pituitary cell line, LbetaT(2), known to express the endogenous LHbeta gene, and into a non-endocrine human embryonic kidney cell line, HEK 293. Basal expression levels of the V-LHbeta promoter constructs were on average 36% higher in LbetaT(2)cells ( P < 0.001; n = 29), and 40% higher in HEK 293 cells ( P < 0.001; n = 16), as compared with the respective wt sequences. Numerous qualitative and quantitative differences were found between the two cell lines in responses of the two promoter sequences to stimulation with 12- O -tetradecanoylphorbol-13-acetate, forskolin, 8-bromo-cAMP, progesterone and gonado- tropin-releasing hormone. In conclusion, the V-LHbeta promoter has higher basal activity, and differs in response to hormonal stimulation, as compared with the wt-LHbeta promoter. The altered promoter function of the V-LHbeta gene provides evidence for differences in regulation of the wt- and V-LHbeta genes, which may contribute to the differences observed in pituitary-gonadal function between carriers of the two LHbeta alleles. The findings also suggest a novel evolutionary mechanism whereby polymorphic changes resulting in altered bioactivity of a gene product may be compensated for by additional mutations in the cognate promoter sequence, changing transcription of the same gene.