Fu-Ping Zhang

ONTOGENY OF FOLLICLE-STIMULATING HORMONE RECEPTOR GENE EXPRESSION IN THE RAT TESTIS AND OVARY

The ontogeny of the follicle-stimulating hormone (FSH) receptor (R) gene expression was studied in the rat testis and ovary between day 12.5 or 14.5 of fetal life (f), respectively, and adulthood. In Northern blots hydbridized with a cRNA probe corresponding to a part of the extracellular domain of the FSHR, specific hybridization to testicular RNA was detected from day f18.5, and to ovarian RNA from postnatal day 7 onwards. The main transcripts in the testis were at all ages 7.0 kb and 2.5 kb in size. In the ovary, the main transcript was always 2.5 kb in size. In order to increase the sensitivity of mRNA detection, the FSHR gene expression was also analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with primer pairs corresponding to the near full-length FSHR mRNA or to its extracellular domain. The specificity of the PCR products was verified by Southern hybridization using a nested 32P-labeled cDNA probe. The results indicated that the expression of the extracellular domain of the FSHR was first detected on day f14.5 in the testis and on day f20.5 in the ovary. The full-length mRNA appeared in both sexes 2 days later, which is in agreement with earlier measurements of appearance of FSHR binding in the rat testis (day f17.5) and ovary (day 3 post partum). In situ hybridization using an antisense cRNA probe for FSHR demonstrated that, as early in development as specific hybridization was detected, it was confined to the Sertoli cells in the testis and to granulosa cells in the ovary. When compared with the developmental onset of the LHR gene expression (our earlier data), a major difference was observed in the ovary; the message encoding the extracellular LHR domain appeared > 10 days earlier than that corresponding to the full-length LHR message. In the case of mRNAs for the testicular LHR, and for FSHR of both sexes, the difference between the developmental appearance of the truncated and full-length RNA forms was only 2 days.

Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures.

The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need.

Sumo-1 Function Is Dispensable in Normal Mouse Development

ABSTRACT To elucidate SUMO-1 functions in vivo, we targeted by homologous recombination the last three exons of the murine Sumo-1 gene. Sumo-1 mRNA abundance was reduced to one-half in heterozygotes and was undetectable in Sumo-1−/− mice, and SUMO-1-conjugated RanGAP1 was detectable in wild-type mouse embryo fibroblasts (MEFs) but not in Sumo-1−/− MEFs, indicating that gene targeting yielded Sumo-1-null mice. Sumo-1 mRNA is expressed in all tissues of wild-type mice, and its abundance is highest in the testis, brain, lungs, and spleen. Sumo-2 and Sumo-3 mRNAs are also expressed in all tissues, but their abundance was not upregulated in Sumo-1-null mice. The development and function of testis are normal in the absence of Sumo-1, and Sumo-1−/− mice of both sexes are viable and fertile. In contrast to a previous report (F. S. Alkuraya et al., Science 313:1751, 2006), we did not observe embryonic or early postnatal demise of Sumo-1-targeted mice; genotypes of embryos and 21-day-old mice were of predicted Mendelian ratios, and there was no defect in lip and palate development in Sumo-1+/− or Sumo-1−/− embryos. The ability of Sumo-1−/− MEFs to differentiate into adipocyte was not different from that of wild-type MEFs. Collectively, our results support the notion that most, if not all, SUMO-1 functions are compensated for in vivo by SUMO-2 and SUMO-3.

The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis

Spermatogenesis is thought to critically depend on the high intratesticular testosterone (T) levels induced by gonadotropic hormones. Strategies for hormonal male contraception are based on disruption of this regulatory mechanism through blockage of gonadotropin secretion. Although exogenous T or T plus progestin treatments efficiently block gonadotropin secretion and suppress testicular T production, only ≈60% of treated Caucasian men reach contraceptive azoospermia. We now report that in luteinizing hormone receptor knockout mice, qualitatively full spermatogenesis, up to elongated spermatids of late stages 13-16, is achieved at the age of 12 months, despite absent luteinizing hormone action and very low intratesticular T (2% of control level). However, postmeiotic spermiogenesis was blocked by the antiandrogen flutamide, indicating a crucial role of the residual low testicular T level in this process. The persistent follicle-stimulating hormone action in luteinizing hormone receptor knockout mice apparently stimulates spermatogenesis up to postmeiotic round spermatids, as observed in gonadotropin-deficient rodent models on follicle-stimulating hormone supplementation. The finding that spermatogenesis is possible without a luteinizing hormone-stimulated high level of intratesticular T contradicts the current dogma. Extrapolated to humans, it may indicate that only total abolition of testicular androgen action will result in consistent azoospermia, which is necessary for effective male contraception.

Developmental and Hormonal Regulation of Leptin Receptor (Ob-R) Messenger Ribonucleic Acid Expression in Rat Testis

Abstract 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 μg/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.

The mouse inhibin α-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice

Abstract Testicular tumorigenesis was observed in transgenic mice expressing the 6-kb mouse inhibin α-subnit promoter/Simian virus 40 T-antigen (SV40 Tag) fusion gene. The tumors were confined to Leydig cells using immunohistochemistry with anti-Tag antibody, specific binding of biotinylated hCG and histochemistry for 3β-hydroxysteroid dehydrogenase. Leydig cell hyperplasia and presence of Tag protein in the testicular interstitial tissue were already evident at 5 and 6.5 days of age, respectively. An immortalized cell line, BLT-1, was established from one testicular tumor. These cells expressed the LH receptor and P450scc mRNAs, and displayed LH-responsive cAMP and progesterone production, and low testosterone production. The cells also specifically bound 125 I-labeled recombinant human LH with high affinity (36 000 binding sites/cell), and the binding was regulated by 8Br-cAMP and hCG. This gonadal tumor model is valuable for further studies on endocrine functions of Leydig cells and their tumorigenesis in vivo and in vitro.

Transgenic and knockout mouse models for the study of luteinizing hormone and luteinizing hormone receptor function

The main functions of luteinizing hormone (LH) are concerned with regulation of gonadal function, and these functions are today well delineated through previous physiological studies. However, novel information of less well-known aspects of actions of this hormone is currently emerging from studies on genetically modified mouse models, with either enhanced or suppressed LH/LH receptor (LHR) function. The novel functions of LH include its role, in specific situations, as promoter of formation and growth of gonadal and extragonadal tumors. Chronically elevated LH levels in transgenic (TG) mice can also induce responses to this hormone in extragonadal tissues. The knockout (KO) mouse for the LHR has elucidated various less well-known details in the function of LH during ontogeny and adult life. Finally, studies on LHR promoter function have revealed that the expression of this gene occurs in age, sex and tissues-specific fashion. The purpose of this brief review is to summarize some of our recent findings upon studies of TG and KO mice with altered function of LH or its receptor.

Fertility in luteinizing hormone receptor-knockout mice after wild-type ovary transplantation demonstrates redundancy of extragonadal luteinizing hormone action

The luteinizing hormone receptor (LHR), mainly expressed in gonads, is essential for normal reproduction. However, numerous recent studies have also demonstrated LHR expression in multiple extragonadal reproductive and nonreproductive tissues. Although some effects of luteinizing hormone (LH) or its agonist, human chorionic gonadotropin, have been shown in extragonadal sites, their physiological significance remains open. In the present study, we have addressed the function of the extragonadal LHR using LHR-KO mice (LuRKO mice), in which the ovaries of prepubertal mice were orthotopically replaced with pieces of WT ovary using similarly transplanted WT mice as controls. Most ovarian transplants attained normal endocrine function in both groups of mice, as demonstrated by normal age at vaginal opening, estrous cycles, and sexual behavior. Both the LuRKO and WT mice repeatedly became pregnant (9/16 vs. 16/20 after first mating; difference not significant) and delivered similarly sized litters, which grew normally after birth, indicating normal lactation. In conclusion, fertility is restored in LuRKO mice by transplantation of WT ovarian tissue. This is achieved in the absence of extragonadal LHR expression, which indicates physiological redundancy for such receptor sites.

The unique exon 10 of the human luteinizing hormone receptor is necessary for expression of the receptor protein at the plasma membrane in the human luteinizing hormone receptor, but deleterious when inserted into the human follicle-stimulating hormone receptor

The LH receptor (LHR) is a member of the family of G protein-coupled seven-times plasma membrane transversing receptors. Its gene consists of 11 exons, the last one encoding the transmembrane and intracellular domains of the receptor. The FSHR, and its gene, resemble structurally those of the LHR, with the exception that the sequences corresponding to exon 10 in LHR are missing in FSHR, which is thus encoded by a total of ten exons. Our recent studies on the marmoset monkey testis LHR cDNA indicated that an 81 bp nucleotide sequence, encoding the complete exon 10 of the LHR gene in other mammalian species, is absent in this species without affecting the LHR function. To study further the role of the exon 10 encoded sequences of the LHR in the gonadotropin receptor function, a deletion of exon 10 from the human LHR (hLHdeltaexon10R), and a chimeric hFSHR with exon 10 from hLHR inserted (hFSHLHexon10R), were constructed in expression vectors. The results presented here demonstrate that 293 cells transfected with the hLHdeltaexon10R display a decrease in the proportion of the receptor binding at the cell surface, compared with cells transfected with wild-type hLHR. However, the cells expressing hLHdeltaexon10R showed similar high affinity binding of [125I]iodo-hCG as those transfected with wild-type hLHR, in either intact cells or their detergent extracts. In addition, cells expressing the hLHdeltaexon10R and wild-type hLHR displayed similar dose-response of cAMP production to hCG stimulation. Cells transfected with chimeric hFSHLHexon10R showed barely detectable [125I]iodo-FSH binding in intact cells compared with those transfected with wild-type hFSHR. The FSH binding detected in cellular detergent extracts displayed 10-fold lower binding activity than wild-type receptors, in spite of similar level of immunoreactive FSHR protein expression in the transfected cells. The hFSHLHexon10R had a modest 5-fold lower binding affinity for FSH as compared with wild-type hFSHR. In conclusion, the present study indicates that the sequences encoding exon 10 of the hLHR are essential for the LHR expression at the plasma membrane, but deleterious for function if inserted into the hFSHR.

Stress-activated miR-21/miR-21* in hepatocytes promotes lipid and glucose metabolic disorders associated with high-fat diet consumption

Objective miR-21 is an oncomir highly upregulated in hepatocellular carcinoma and in early stages of liver diseases characterised by the presence of steatosis. Whether upregulation of miR-21 contributes to hepatic metabolic disorders and their progression towards cancer is unknown. This study aims at investigating the role of miR-21/miR-21* in early stages of metabolic liver disorders associated with diet-induced obesity (DIO). Design Constitutive miR-21/miR-21* knockout (miR21KO) and liver-specific miR-21/miR-21* knockout (LImiR21KO) mice were generated. Mice were then fed with high-fat diet (HFD) and alterations of the lipid and glucose metabolism were investigated. Serum and ex vivo explanted liver tissue were analysed. Results Under normal breeding conditions and standard diet, miR-21/miR-21* deletion in mice was not associated with any detectable phenotypic alterations. However, when mice were challenged with an obesogenic diet, glucose intolerance, steatosis and adiposity were improved in mice lacking miR-21/miR-21*. Deletion of miR-21/miR-21* specifically in hepatocytes led to similar improvements in mice fed an HFD, indicating a crucial role for hepatic miR-21/miR-21* in metabolic disorders associated with DIO. Further molecular analyses demonstrated that miR-21/miR-21* deletion in hepatocytes increases insulin sensitivity and modulates the expression of multiple key metabolic transcription factors involved in fatty acid uptake, de novo lipogenesis, gluconeogenesis and glucose output. Conclusions Hepatic miR-21/miR-21* deficiency prevents glucose intolerance and steatosis in mice fed an obesogenic diet by altering the expression of several master metabolic regulators. This study points out miR-21/miR-21* as a potential therapeutic target for non-alcoholic fatty liver disease and the metabolic syndrome.