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.

[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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Direct luteinizing hormone action triggers adrenocortical tumorigenesis in castrated mice transgenic for the murine inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene.

Transgenic (TG) mice, expressing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin alpha-subunit promoter (inh alpha p), develop gonadal tumors of granulosa/theca or Leydig cell origin. We showed previously that adrenocortical tumors develop if the TG mice are gonadectomized but never develop in intact animals. However, if functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding the TG mice into the hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumors appeared. Since the most obvious difference between the gonadectomized and GnRH-antagonist-treated or Tag/hpg double mutant mice is the elevated gonadotropin secretion in the first group, we examined whether the adrenal tumorigenesis would be gonadotropin-dependent. Surprisingly, both the adrenal tumors and a cell line (C alpha 1) derived from one of them expressed highly functional LH receptors (LHR), as assessed by Northern hybridization, immunocytochemistry, ligand binding, and human CG (hCG)-stimulated cAMP and steroid production. No FSH receptor expression was found in the adrenal tumors by RT-PCR. hCG treatment of the C alpha 1 cells stimulated their proliferation, as measured by [3H]thymidine incorporation. This effect was related to hCG-stimulated steroidogenesis since progesterone, testosterone, and estradiol, at physiological concentrations, also stimulated the C alpha 1 cell proliferation. Different adrenocortical cells expressed initially LHR and Tag, whereas both were highly expressed in the tumor cells. In conclusion, the high level of functional LHR in the adrenal tumors indicates that this receptor can function as tumor promoter when ectopically expressed and stimulated by the ligand hormone.

Persistent expression of a truncated form of the luteinizing hormone receptor messenger ribonucleic acid in the rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

To correlate the process of ethylene dimethane sulfonate (EDS)-induced disappearance and repopulation of Leydig cells with LH receptor (LHR) expression, testicular messenger RNA (mRNA) and binding of LHR were analyzed in male rats, 5, 15, 20, and 40 days after treatment with 75 mg EDS/kg BW. Five and 15 days after EDS treatment, the serum testosterone level was reduced by 90% (P < 0.01) and testicular [125I]iodo-hCG binding was nearly undetectable (P < 0.01). Multiple splice variants of the LHR mRNA, with sizes of 6.8, 4.2, 2.7, and 1.8 kilobases, were detected in control testes upon Northern hybridization. Interestingly, 5 and 15 days after EDS injection, only the 1.8-kilobase band, previously reported to correspond to a truncated form of the LHR mRNA and encoding its extracellular part, remained, whereas the other mRNA species disappeared. On days 20 and 40 after EDS treatment, the pattern of hybridization gradually returned to that resembling the control pattern. To increase the sensitivity of mRNA detection, testicular RNA was reverse transcribed and amplified by polymerase chain reaction, using primers complementary to various parts of the LHR complementary DNA. The specificity of the complementary DNAs generated was verified by Southern hybridization with nested oligonucleotide primers. Five and 15 days after EDS treatment, only truncated mRNA forms, encoding regions of the extracellular part of the LHR, could be amplified. At 20 and 40 days, the pattern of amplification was similar to that in control testes, with amplification of the whole coding sequence. In situ hybridization was performed on day 5 after EDS treatment, when the interstitial space was devoid of morphologically discernible Leydig cells. An antisense complementary RNA probe, corresponding to the extracellular domain of the receptor, hybridized in the interstitial space to apparent precursor Leydig cells. Taken together, these results strongly suggest that the precursor Leydig cells, resistant to the cytotoxic action of EDS, express truncated forms of the LHR mRNA in the early stages of their differentiation. The full-length mRNA of LHR gradually appears when the functional Leydig cells recover and attain their differentiated functions. These data are analogous with our previous findings on testicular ontogeny; the fetal Leydig cell precursors constitutively express a truncated form of the LHR gene as well, and a similar change occurs in its alternative splicing during testicular maturation. Hence, the truncated form of the LHR mRNA is an early sign of Leydig cell differentiation, whether it occurs during ontogeny or in adulthood upon recovery from cytotoxic treatment.

Ontogeny of luteinizing hormone receptor gene expression in the rat testis.

The ontogeny of expression of the LH receptor (LHR) gene was studied in rat testis between day 12.5 of fetal life and adulthood. Specific hybridization of testicular mRNA with a LHR cRNA probe encoding the extracellular domain of the receptor was found from day 16.5 of fetal life onward in Northern hybridization. Transcripts of 6.8, 4.2, and 2.7 kilobases were present at all ages, and a 1.8-kilobase species was present mainly in the adult testes. Hybridization was most intensive in day 21.5 fetuses, decreased after birth, and increased again by adulthood. The LHR mRNA was also analyzed by the reverse transcriptase-polymerase chain reaction technique, with primers multiplying either the full-length LHR mRNA or its extracellular domain. The specificity of the DNA species generated was verified by Southern hybridization using a nested 32P-labeled oligonucleotide. The results indicated that a truncated mRNA form, encoding the extracellular part of LHR, appears 1 day before the full-length LHR mRNA, i.e. on fetal days 14.5 and 15.5, respectively. This is in striking contrast to the rat fetal ovary, in which a difference of more than 10 days is found in the appearance of these two LHR mRNAs (17.5 days of fetal and 7 days of postnatal age, respectively). The appearance of the full-length LHR mRNA coincides in both sexes with the developmental onset of LHR binding observed in earlier studies. In situ hybridization using an antisense cRNA probe demonstrated that the LHR mRNA was confined to Leydig cells at all fetal and postnatal ages studied. In conclusion, there is good correlation in the developing rat testes between the onset of LHR gene expression and LHR binding, as observed in earlier studies. The findings in the fetal testis are at striking variance with those in the ovary, which starts expressing the extracellular domain of the LHR mRNA at roughly the same age as the testis. However, the appearance of full-length LHR mRNA and the functional receptor are delayed until day 7 postpartum.

Novel expression of luteinizing hormone subunit genes in the rat testis.

The two gonadotropins, LH and FSH, are thought to be synthesized and secreted solely by the anterior pituitary. We present here evidence for expression of the LH beta and common alpha-subunit (C alpha) genes in the rat testis. The LH beta and C alpha-subunit messenger RNAs (mRNAs) were detected by reverse transcriptase-polymerase chain reaction in the rat testis and pituitary with primer pairs producing 247- and 199-base pair complementary DNA (cDNA) fragments, corresponding to nucleotides 154-400 of LH beta and nucleotides 250-448 of C alpha cDNA, respectively. The specificity of the cDNA species generated was verified by Southern hybridization using nested [32P]cDNA or oligonucleotide probes, and identity with the published rat LH beta and C alpha-subunit gene structures was determined by sequencing. The mRNA bands with specific hybridization to complementary RNA (cRNA) probes corresponding to nucleotides 154-368 of the rat LH beta cDNA and nucleotides 250-448 of the rat C alpha cDNA were found in the rat pituitary and testis by Northern hybridization. The major C alpha mRNA had a size of 0.8 kilobases (kb) in the pituitary and testis. The major LH beta transcripts were 0.8 and 2.7 kb in the pituitary and testis, respectively. To further characterize the larger testicular LH beta-subunit transcript, rapid amplification of the 3'-end of cDNA (3'-RACE) was performed using an oligo(deoxythymidine-17) adapter and a specific 5'-primer. Southern hybridization of the 3'-RACE product of rat testicular RNA with a LH beta [32P]cDNA probe had the same size as the 3'-RACE product of pituitary RNA. The pituitary and testicular RNAs were then cut into two segments using oligonucleotide-directed ribonuclease H digestion and subjected to Northern hybridization using a cRNA probe specific to the 5'-end segment. The digested 5'-end segments of the pituitary and testicular mRNAs were 0.4 and 2.3 kb, respectively, indicating that the testicular LH beta mRNA has a 1.9-kb 5'-extension, compared to the cognate pituitary mRNA. This was further verified by Northern hybridization using a cRNA probe corresponding to nucleotides -790 to -10 upstream of the pituitary initiation site of LH beta gene transcription. Specific hybridization of a 2.7-kb mRNA transcript was found in the rat testis, but none in the pituitary. Hence, the 3'-end polyadenalytion site of the LH beta mRNA is the same in rat pituitary and testis, and the different transcript sizes are due to a difference at the 5'-end.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species.

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testicular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) containing the complete amino acid-coding sequence (676 amino acids). Northern hybridization analysis of monkey testicular RNA, using a complementary RNA probe corresponding to the full-length cDNA, demonstrated major transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, and 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a striking feature, i.e. the absence of an 81-bp nucleotide sequence corresponding to exon 10, present in the LHR cDNAs of all other species studied to date. The monkey LHR cDNA displayed 83-94% overall sequence homology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridization of monkey genomic DNA using a human exon 10 probe demonstrated its presence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA showed similar high affinity (Kd = 0.25 nmol/liter) of [125I]iodo-hCG binding as those transfected with human LHR cDNA (Kd = 0.20 nmol/liter). The cells expressing the recombinant monkey and human LHR displayed similar responses of extracellular cAMP and inositol trisphosphate to hCG. In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.

Molecular mechanisms of reappearance of luteinizing hormone receptor expression and function in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.

Considering the major role of LH in the control of Leydig cell (LC) development and function, we aimed to characterize further the pattern of LH receptor (LHR) expression in two experimental paradigms: the rat treated with ethylene dimethane sulfonate (EDS), in which the selective destruction of preexisting mature LCs induces the proliferation and differentiation of newly formed LCs, a process that takes place in the presence of high levels of gonadotropins; and the EDS-rat treated with a high dose of testosterone (EDS + T), in which the LH secretion is suppressed, and consequently LC development after EDS arrested. In EDS rats, serum T was suppressed and testicular LHR binding became undetectable on days 5 and 15 after treatment. The pattern of LHR messenger RNA (mRNA) expression was profoundly modified: only one of the splice variants [1.8-kilobase (kb)] persisted, whereas the others disappeared. On days 20 and 45 after EDS, along with LC repopulation, serum T and LHR binding recovered, and the pattern of LHR mRNA expression gradually returned to that resembling controls. In EDS + T rats, a similar drop in testicular LHR binding and change in the pattern of LHR mRNA expression was detected on days 5 and 15 after treatment. However, on days 20 and 45, no recovery either in LHR binding or in expression of the longer LHR mRNA splice variants was observed, showing that LH is needed to induce LHR expression in repopulating LCs, at least to a quantitatively significant level. To gain further insight into the mechanism(s) by which LH acts on LC precursors, the translational status of the 1.8-kb LHR transcript, persistently expressed after EDS, was analyzed and compared with that of the 6.8-kb message. In polysome distribution analysis of total testicular RNA, the 6.8-kb LHR message was highly associated with polysomes, whereas the 1.8-kb variant was mainly localized to prepolysomal fractions, both in control and EDS testes, thus predicting lower translational efficiency. In addition, considering that only LCs express LHRs in the testis, the time course of the reappearance of functional receptors was mapped by evaluating testicular responsiveness to human recombinant LH in vitro. No response to LH stimulation was detected 5 days after EDS. However, cAMP response to LH was observed on days 15 and 20, regardless of the presence of high (EDS) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of functional LHRs, qualitatively, can take place in the absence of measurable LH levels. In EDS-treated rats, the appearance of the cAMP response coincided with those of pregnenolone, progesterone, and T. In contrast, no LH-induced steroid release was observed in EDS + T rats, indicating that steroidogenic response in developing LC requires LH priming. In conclusion, the appearance of functional LHRs, at a low level of expression, in LC precursors is an LH-independent developmental event, essential for the subsequent LH-dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results cast doubt on a major functional role of the truncated (1.8-kb) form of LHR mRNA, which persists after EDS at a high level of expression, in the early Leydig cell precursors.

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.