Global, Regional, and Country-Specific Lifetime Risks of Stroke, 1990 and 2016.

BACKGROUND: The lifetime risk of stroke has been calculated in a limited number of selected populations. We sought to estimate the lifetime risk of stroke at the regional, country, and global level using data from a comprehensive study of the prevalence of major diseases. METHODS: We used the Global Burden of Disease (GBD) Study 2016 estimates of stroke incidence and the competing risks of death from any cause other than stroke to calculate the cumulative lifetime risks of first stroke, ischemic stroke, or hemorrhagic stroke among adults 25 years of age or older. Estimates of the lifetime risks in the years 1990 and 2016 were compared. Countries were categorized into quintiles of the sociodemographic index (SDI) used in the GBD Study, and the risks were compared across quintiles. Comparisons were made with the use of point estimates and uncertainty intervals representing the 2.5th and 97.5th percentiles around the estimate. RESULTS: The estimated global lifetime risk of stroke from the age of 25 years onward was 24.9% (95% uncertainty interval, 23.5 to 26.2); the risk among men was 24.7% (95% uncertainty interval, 23.3 to 26.0), and the risk among women was 25.1% (95% uncertainty interval, 23.7 to 26.5). The risk of ischemic stroke was 18.3%, and the risk of hemorrhagic stroke was 8.2%. In high-SDI, high-middle-SDI, and low-SDI countries, the estimated lifetime risk of stroke was 23.5%, 31.1% (highest risk), and 13.2% (lowest risk), respectively; the 95% uncertainty intervals did not overlap between these categories. The highest estimated lifetime risks of stroke according to GBD region were in East Asia (38.8%), Central Europe (31.7%), and Eastern Europe (31.6%), and the lowest risk was in eastern sub-Saharan Africa (11.8%). The mean global lifetime risk of stroke increased from 22.8% in 1990 to 24.9% in 2016, a relative increase of 8.9% (95% uncertainty interval, 6.2 to 11.5); the competing risk of death from any cause other than stroke was considered in this calculation. CONCLUSIONS: In 2016, the global lifetime risk of stroke from the age of 25 years onward was approximately 25% among both men and women. There was geographic variation in the lifetime risk of stroke, with the highest risks in East Asia, Central Europe, and Eastern Europe. (Funded by the Bill and Melinda Gates Foundation.).

Health Effects of Overweight and Obesity in 195 Countries over 25 Years.

BACKGROUND: Although the rising pandemic of obesity has received major attention in many countries, the effects of this attention on trends and the disease burden of obesity remain uncertain. METHODS: We analyzed data from 68.5 million persons to assess the trends in the prevalence of overweight and obesity among children and adults between 1980 and 2015. Using the Global Burden of Disease study data and methods, we also quantified the burden of disease related to high body-mass index (BMI), according to age, sex, cause, and BMI in 195 countries between 1990 and 2015. RESULTS: In 2015, a total of 107.7 million children and 603.7 million adults were obese. Since 1980, the prevalence of obesity has doubled in more than 70 countries and has continuously increased in most other countries. Although the prevalence of obesity among children has been lower than that among adults, the rate of increase in childhood obesity in many countries has been greater than the rate of increase in adult obesity. High BMI accounted for 4.0 million deaths globally, nearly 40% of which occurred in persons who were not obese. More than two thirds of deaths related to high BMI were due to cardiovascular disease. The disease burden related to high BMI has increased since 1990; however, the rate of this increase has been attenuated owing to decreases in underlying rates of death from cardiovascular disease. CONCLUSIONS: The rapid increase in the prevalence and disease burden of elevated BMI highlights the need for continued focus on surveillance of BMI and identification, implementation, and evaluation of evidence-based interventions to address this problem. (Funded by the Bill and Melinda Gates Foundation.).

Training alters the distribution of perilipin proteins in muscle following acute free fatty acid exposure.

KEY POINTS: The lipid droplet (LD)-associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, although whether the abundance and association of the PLIN proteins with LDs is related to the diverse lipid storage in muscle between trained and sedentary individuals is unknown. We show that lipid infusion augments IMTG content in type I fibres of both trained and sedentary individuals. Most importantly, despite there being no change in PLIN protein content, lipid infusion did increase the number of LDs connected with PLIN proteins in trained individuals only. We conclude that trained individuals are able to redistribute the pre-existing pool of PLIN proteins to an expanded LD pool during lipid infusion and, via this adaptation, may support the storage of fatty acids in IMTG. ABSTRACT: Because the lipid droplet (LD)-associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, we investigated the hypothesis that differential protein content of PLINs and their distribution with LDs may be linked to the diverse lipid storage in muscle between trained and sedentary individuals. Trained (n = 11) and sedentary (n = 10) subjects, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed using confocal immunofluorescence microscopy for fibre type-specific IMTG content and PLIN associations with LDs. In both groups, lipid infusion increased IMTG content in type I fibres (trained: +62%, sedentary: +79%; P < 0.05) but did not affect PLIN protein content. At baseline, PLIN2 (+65%), PLIN3 (+105%) and PLIN5 (+53%; all P < 0.05) protein content was higher in trained compared to sedentary individuals. In trained individuals, lipid infusion increased the number of LDs associated with PLIN2 (+27%), PLIN3 (+73%) and PLIN5 (+40%; all P < 0.05) in type I fibres. By contrast, in sedentary individuals, lipid infusion only increased the number of LDs not associated with PLIN proteins. Acute free fatty acid elevation therefore induces a redistribution of PLIN proteins to an expanded LD pool in trained individuals only and this may be part of the mechanism that enables fatty acids to be stored in IMTG.

First Report of Resistance to QoI Fungicides in Alternaria alternata Isolates from Potato in South Africa.

Since the first report of brown spot of potatoes (Solanum tuberosum) caused by Alternaria alternata (Fr.) Keissl in South Africa (3), disease intensity has steadily increased. No fungicides are registered for control of brown spot of potatoes in South Africa but growers attempt to control the disease with products registered for early blight, which include various QoI fungicides. Failure to control brown spot with QoI fungicides led to an investigation on putative development of resistance among A. alternata populations. In the summer of 2012, diseased leaves were collected from five potato growing regions. Isolations were made from the margin of brown spot lesions by plating surface disinfested tissue on V8 agar medium (200 ml V8 juice, 3 g CaCO3, 20 g agar). Plates were incubated at 25°C in darkness for 7 days, purified, and single-spore cultures transferred to fresh potato dextrose agar (PDA) plates. Identity of isolates was confirmed using conidial morphology and PCR amplification using species-specific primers AAF2 and AAR3 (1). Eight A. alternata isolates (PPRI 13607, 13608, 13609, 13610, 13611, 13612, 13613, and 13614) were obtained and screened for sensitivity to azoxystrobin in vitro by evaluating relative conidial germination on media amended with 0, 1.0, 2.5, 5.0, 10, 25, 50, 75, 85, and 100 µg of azoxystrobin per ml of media. The dose effect of the fungicide on germination and the EC50 of each isolate were computed using the probit analysis. Isolates were subjected to DNA extraction and the partial cytochrome b (cyt b) was amplified using primer pair CBF1 and CBR2 (2). PCR products were transformed into DH5α competent cells using a pGEM-T Easy vector. Both strands of the cloned fragments were sequenced using primers T7 and SP6 (4). Isolates PPRI 13611 and 13614 had low EC50 values of 0.11 and 0.23 µg/ml, respectively, and a mean EC50 of 0.17 µg/ml, showing their relative sensitivity to azoxystrobin. The other six isolates had EC50 values ranging from 51.88 to 114.92 µg/ml, and a mean EC50 of 71.60 µg/ml, showing their resistance to azoxystrobin. Sequence analysis of the partial cyt b gene showed strong association of resistance in isolates PPRI 13607, 13608, 13609, 13610, 13612, and 13613 to a base substitution resulting in an amino acid substitution at position 143 (G143A). Isolates PPRI 13611 and 13614 did not exhibit this mutation. Although resistance has been reported on other crops where QoI fungicides, including azoxystrobin, have been used to control different pathogens, this is the first report of resistance to a QoI fungicide in field isolates of A. alternata from potatoes in South Africa. Identification of resistance will help to explain failure to control this disease using QoI fungicides. Further monitoring of resistance to azoxystrobin and other QoI fungicides is warranted. References: (1) P. Konstantinova et al. Mycol. Res. 106:23, 2002. (2) Z. Ma et al. Pesticide Biochem. Phys. 77:66, 2003. (3) J. van der Waals et al. Plant Dis. 95:363, 2011. (4) E. Youssef et al. DNA Seq. 11:541, 2001.

EEG rhythmic and arrhythmic spectral components and functional connectivity at resting state may predict the development of synucleinopathies in idiopathic REM sleep behavior disorder.

STUDY OBJECTIVES: Idiopathic/isolated REM-sleep behavior disorder (iRBD) often precedes the onset of synucleinopathies. Here, we investigated whether baseline resting-state EEG advanced spectral power and functional connectivity differ between iRBD patients who converted towards a synucleinopathy at follow-up and those who did not. METHODS: Eighty-one participants with iRBD (66.89±6.91 years) underwent a baseline resting-state EEG recording, a neuropsychological assessment and a neurological examination. We estimated EEG power spectral density using standard analyses and derived spectral estimates of rhythmic and arrhythmic components. Global and pairwise EEG functional connectivity analyses were computed using the weighted phase-lag index (wPLI). Pixel-based permutation tests were used to compare groups. RESULTS: After a mean follow-up of 5.01±2.76 years, 34 patients were diagnosed with a synucleinopathy (67.81±7.34 years) and 47 remained disease-free (65.53±7.09 years). Among patients who converted, 22 were diagnosed with Parkinson's disease and 12 with dementia with Lewy bodies. As compared to patients who did not convert, patients who converted exhibited at baseline higher relative theta standard power, steeper slopes of the arrhythmic component and higher theta rhythmic power mostly in occipital regions. Furthermore, patients who converted showed higher beta global wPLI but lower alpha wPLI between left temporal and occipital regions. CONCLUSION: Analyses of resting-state EEG rhythmic and arrhythmic components and functional connectivity suggest an imbalanced excitatory-to-inhibitory activity within large-scale networks, which is associated with later development of a synucleinopathy in iRBD patients.

Effects of weight loss and exercise on insulin resistance, and intramyocellular triacylglycerol, diacylglycerol and ceramide.

AIMS/HYPOTHESIS: Intramyocellular lipids, including diacylglycerol (DAG) and ceramides, have been linked to insulin resistance. This randomised repeated-measures study examined the effects of diet-induced weight loss (DIWL) and aerobic exercise (EX) on insulin sensitivity and intramyocellular triacylglycerol (IMTG), DAG and ceramide. METHODS: Sixteen overweight to obese adults (BMI 30.6 ± 0.8; 67.2 ± 4.0 years of age) with either impaired fasting glucose, or impaired glucose tolerance completed one of two lifestyle interventions: DIWL (n = 8) or EX (n = 8). Insulin sensitivity was determined using hyperinsulinaemic-euglycaemic clamps. Intramyocellular lipids were measured in muscle biopsies using histochemistry and tandem mass spectrometry. RESULTS: Insulin sensitivity was improved with DIWL (20.6 ± 4.7%) and EX (19.2 ± 12.9%). Body weight and body fat were decreased by both interventions, with greater decreases in DIWL compared with EX. Muscle glycogen, IMTG content and oxidative capacity were all significantly (p < 0.05) decreased with DIWL and increased with EX. There were decreases in DAG with DIWL (-12.4 ± 14.6%) and EX (-40.9 ± 12.0%). Ceramide decreased with EX (-33.7 ± 11.2%), but not with DIWL. Dihydroceramide was decreased with both interventions. Sphingosine was decreased only with EX. Changes in total DAG, total ceramides and other sphingolipids did not correlate with changes in glucose disposal. Stearoyl-coenzyme A desaturase 1 (SCD1) content was decreased with DIWL (-19.5 ± 8.5%, p < 0.05), but increased with EX (19.6 ± 7.4%, p < 0.05). Diacylglycerol acyltransferase 1 (DGAT1) was unchanged with the interventions. CONCLUSIONS/INTERPRETATION: Diet-induced weight loss and exercise training both improved insulin resistance and decreased DAG, while only exercise decreased ceramides, despite the interventions having different effects on IMTG. These alterations may be mediated through differential changes in skeletal muscle capacity for oxidation and triacylglycerol synthesis. TRIAL REGISTRATION: ClinicalTrials.gov NCT00766298.

Phenotypic variability among patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome homozygous for the delF188 mutation in SLC25A15.

BACKGROUND: Hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome (OMIM 238970) is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15. To date, 22 different mutations of the SLC25A15 gene have been described in 49 patients belonging to 31 unrelated families. OBJECTIVE: To further delineate the phenotypic spectrum of HHH syndrome from a description of a genetically homogeneous cohort of patients and identify prognostic factors based on long-term follow-up. METHODS: Sixteen French-Canadian patients were retrospectively and prospectively clinically assessed. RESULTS: Owing to a founder effect, 15 of the 16 patients were homozygous for the F188del mutation in the SLC25A15 gene. The main clinical features at presentation were liver dysfunction (6/16) and neurological disease (9/16), including chronic neurological symptoms (6/9) and acute encephalopathy (3/9). Hyperammonaemia was not constant and usually mild and uncommon after start of treatment. Long-term follow-up showed that variable intellectual impairment and lower limb spasticity often occur, together or separately, with no obvious relationship to age at diagnosis and compliance with treatment. CONCLUSION: We report the largest known cohort to date of patients with HHH syndrome. A similar range of severity occurred in the clinical course and outcome of patients homozygous for delF188 and in the 33 other reported patients compiled from the literature. The poor clinical outcome of some patients with HHH syndrome despite early treatment and repeatedly normal plasma ammonia levels emphasises the need to better understand the pathophysiology and to reconsider the therapeutic goals for HHH.

Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.

Mental activity and referential activity of beginning therapists: a construct validity study of the Countertransference Rating System (CRS).

This study investigates the construct validity of a revised version of the Countertransference Rating System (CRS) by means of convergence with Referential Activity (RA). The CRS operationalizes three mental activity dimensions (rational-objective, reactive, and reflective) as processes of transformation of countertransferential contents elicited in a therapist by a patient's object-relations units. The participants were 36 novice psychotherapists who shared their spontaneous reactions toward parental descriptions provided by conduct-disordered male adolescent patients. Globally, the reflective dimension was positively correlated with RA, whereas the other two dimensions--rational-objective and reactive--showed no association. Dominant categories within each dimension displayed patterns of correlation with RA that are consistent with the constructs. These results are discussed with reference to potential impact on intervention and training.

Enzymatic characterization of arginine esterase from dog seminal plasma.

Previously purified arginine esterase from dog seminal plasma was characterized enzymatically. The enzyme was found to have a rather narrow specificity for arginine esters, much less for lysine esters and was practically devoid of activity towards tyrosine esters, casein, albumin and azocoll. It had a broad optimum pH between 8 and 9. It presented no kallikrein-like activities either in the blood pressure test in dog or in the rat uterus contraction test. It was inhibited by bovine pancreas trypsin inhibitor, aprotinin, phenylalanylprolyl arginine chloromethyl ketone, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate and leupeptin, but not by soybean trypsin inhibitor, tosyllysine chloromethyl ketone, tosylamide-2-phenylethyl chloromethyl ketone, iodoacetamide, Triton X-100 and EDTA. Experiments involving incubation of prostatic cytosol with purified arginine esterase showed that actin was the only important prostatic protein that was extensively hydrolyzed by this enzyme. It is not known presently whether the hydrolysis of actin is related to a true physiological function of the enzyme and whether actin and arginine esterase ever come into contact with each other in vivo. These properties indicate that arginine esterase from dog seminal plasma is different from other known proteinases including classical kallikreins, although it presents many similarities with this class of enzyme.

Characterization of rhesus monkey prostate specific antigen cDNA.

Using a RT-PCR approach, we obtained two overlapping cDNA clones containing the entire 1.5 kb sequence of rhesus monkey prostate specific antigen (rmPSA). The sequence obtained revealed an open reading frame of 261 amino acids. One potential N-glycosylation site was identified at Asn-78. The calculated molecular mass for the unglycosylated mature protein was 26,147 Da. Extensive amino acid homology (89%) was observed between rmPSA and its human counterpart. These results demonstrate that rhesus monkey and man prostate share a major biochemical component, and suggest that this animal species might be useful to answer specific questions related to human prostatic function and pathology.

Purification of enzymatically active kallikrein hK2 from human seminal plasma.

Kallikrein hK2 is a member of the human glandular kallikrein family which includes prostate-specific antigen (PSA) and pancreatic-renal kallikrein. The purpose of this work was to isolate and characterize for the first time the enzymatically active form of the hK2 protein starting from the PCI-hK2 complex isolated from human seminal plasma (Deperthes, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Hébert, J., Lazure, C. and Dubé, J.Y. (1995) Biochim. Biophys. Acta 1245, 311-316). That complex was dissociated by an incubation at alkaline pH and final purification was achieved by C-18 reverse phase HPLC. The purified material contained a 27 kDa band by SDS gel electrophoresis and had the expected NH2-terminal amino acid sequence of hK2. It hydrolyzed synthetic chromogenic substrates containing esters of lysine and arginine but not of phenylalanine. Furthermore, hK2 formed molecular complexes with alpha 2 -antiplasmin, alpha 1-antichymotrypsin, antithrombin III and alpha 2-macroglobulin but not with alpha 1-antitrypsin. In conlusion, the new findings of the present paper are that the PCI-hK2 complex can be dissociated by mild procedures, that the free hK2 protein can be purified thereafter by standard HPLC procedures, that the recovered free hK2 is a trypsin-like enzyme and that it can form molecular complexes with many of the major serum proteinase inhibitors.

Identification of glandular kallikrein in dog pancreas and determination of its tissue distribution.

In order to establish a formal link between previously purified canine urinary kallikrein and dog pancreatic kallikrein whose cDNA sequence has recently been published, we have isolated the pancreatic kallikrein from that animal species. Pancreatic cytosol proteins were sequentially subjected to chromatography on DEAE-Sepharose CL-6B and Concanavalin A-Sepharose, to an autolysis step and finally to two-dimensional gel electrophoresis. Kallikrein immunoreactive spots were identified with an antibody directed against canine urinary kallikrein. These proteins were isolated after electroblotting and the amino acid sequence of their NH2-terminal portion was determined by microsequencing. The sequence was found to be identical to the one deduced from pancreatic kallikrein cDNA. Using the same antibody and immunohistochemical procedures, kallikrein was found to be present in the pancreas, the salivary glands, the kidney, the colon, the lungs and the testis. These results thus confirm the molecular nature of a glandular kallikrein in the canine species.

Characterization of canine pancreas kallikrein cDNA.

Using a combination of primer extension and RT-PCR, the cDNA encoding a canine tissue kallikrein expressed in the pancreas was cloned and sequenced. The cloned 0.85 kbp cDNA contained a complete open reading frame encoding a polypeptide of 261 amino acids. The calculated molecular mass of the processed, unglycosylated, 237 amino acid protein was 26,428 Da. Its mRNA was expressed at high levels in the pancreas, kidney and submaxillary gland. The sequence of the encoded protein was highly homologous with canine prostatic arginine esterase (66%) and human renal/pancreatic kallikrein (74%). Therefore, the cloned cDNA encoded a previously uncharacterized canine kallikrein enzyme which was named dog renal/pancreatic kallikrein or dK2 according to the new nomenclature for kallikrein gene family members. Because of its specific pattern of tissue expression and the presence of all the amino acid residues necessary for kininogenase activity, we suggest that dK2 is the canine true tissue kallikrein.

Human kallikrein hK2 has low kininogenase activity while prostate-specific antigen (hK3) has none.

In the present paper, we determined the kinin-releasing activity of human prostatic kallikrein hK2 and compared it to one of the kallikreins hK1 and prostate specific antigen (hK3). Kinin-like substances active on the rabbit jugular vein were progressively produced when nanomolar concentrations of hK2 were incubated with heated plasma. However in these experiments, hK1 appeared much more potent than hK2 while hK3 was totally inactive. When hK2 was incubated with purified high molecular weight kininogen, several peptides were generated as shown by the analysis on C18 reverse-phase HPLC. Kinin activity was localized exclusively in a small peak having an elution time identical to that of bradykinin while the only important peak obtained with hK1 corresponded to Lys-bradykinin. Finally, the rate of kinin production of hK2 was found to be more than a thousandfold lower than that of hK1. These experiments show that kallikreins hK2 has only a low kininogenase activity. However, it is not excluded that some of the peptides produced by hK2 action could have other types of biological activity.

Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma.

To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.

Epstein-Barr virus polymorphic B-cell lymphoma associated with leukemia and with congenital immunodeficiencies.

Polymorphic B-cell lymphoma seen in four patients with congenital immunodeficiencies and in two patients with leukemia receiving chemotherapy was associated with the Epstein-Barr virus (EBV). The tumors had characteristic histologic features: they were polymorphic consisting of a mixture of lymphoblasts and differentiated cells including plasma cells, and areas of hemorrhagic necrosis were prominent. The tumors were either polyclonal, monoclonal, or multiclonal. Patients with congenital immunodeficiencies who developed these tumors died despite radiotherapy, corticosteroids plus acyclovir, or a combination of intravenous (IV) immunoglobulins and alpha 2 interferon. Patients with leukemia recovered when immunosuppressive drugs were discontinued and leukemia has not recurred over a period of 2 and 4 years, respectively, in the two patients.

The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes.

We have taken advantage of the sequence relationships among the bone morphogenetic proteins (BMPs) to identify the mouse Bmp15 and human BMP15 genes. The 392-amino acid prepropeptides encoded by these BMP genes exhibit significant homology to each other, although the 70% identity observed between the 125-amino acid mature peptides is considerably lower than that seen in comparisons of other mouse and human orthologs. Both genes share a common structural organization and encode mature peptides that lack the cysteine residue normally involved in the formation of a covalent dimer. In addition, mouse Bmp15 and human BMP15 map to conserved syntenic regions on the X chromosome. We demonstrate, through a combination of Northern blot and in situ hybridization analyses, that mouse Bmp15 is expressed specifically in the oocyte beginning at the one-layer primary follicle stage and continuing through ovulation. Interestingly, BMP-15 is most closely related to and shares a coincident expression pattern with the mouse growth/differentiation factor 9 (GDF-9) gene that is essential for female fertility. Our findings will be important for defining the role of BMP-15 in follicular development.

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.