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BACKGROUND: Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity. RESULTS: To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches. CONCLUSIONS: This bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites.
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Bactérias , Bexiga Urinária , Adulto , Humanos , Feminino , Bexiga Urinária/microbiologia , Filogenia , RNA Ribossômico 16S/genética , Bactérias/genética , Escherichia coli/genética , CatalogaçãoRESUMO
BACKGROUND: Enterococcus faecalis is the most commonly isolated enterococcal species in clinical infection. This bacterium is notorious for its ability to share genetic content within and outside of its species. With this increased proficiency for horizontal gene transfer, tremendous genomic diversity within this species has been identified. Many researchers have hypothesized E. faecalis exhibits niche adaptation to establish infections or colonize various parts of the human body. Here, we hypothesize that E. faecalis strains isolated from the human bladder will carry unique genomic content compared to clinical strains isolated from other sources. RESULTS: This analysis includes comparison of 111 E. faecalis genomes isolated from bladder, urogenital, blood, and fecal samples. Phylogenomic comparison shows no association between isolation source and lineage; however, accessory genome comparison differentiates blood and bladder genomes. Further gene enrichment analysis identifies gene functions, virulence factors, antibiotic resistance genes, and plasmid-associated genes that are enriched or rare in bladder genomes compared to urogenital, blood, and fecal genomes. Using these findings as training data and 682 publicly available genomes as test data, machine learning classifiers successfully distinguished between bladder and non-bladder strains with high accuracy. Genes identified as important for this differentiation were often related to transposable elements and phage, including 3 prophage species found almost exclusively in bladder and urogenital genomes. CONCLUSIONS: E. faecalis strains isolated from the bladder contain unique genomic content when compared to strains isolated from other body sites. This genomic diversity is most likely due to horizontal gene transfer, as evidenced by lack of phylogenomic clustering and enrichment of transposable elements and prophages. Investigation into how these enriched genes influence host-microbe interactions may elucidate gene functions required for successful bladder colonization and disease establishment.
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Enterococcus faecalis , Genoma Bacteriano , Humanos , Enterococcus faecalis/genética , Elementos de DNA Transponíveis/genética , Bexiga Urinária , Genômica , Antibacterianos , Prófagos/genéticaRESUMO
Average nucleotide identity analysis, based on whole genome sequences of 115 strains previously identified as Aerococcus urinae, an emerging uropathogen, discriminates at least six unique genomic taxa. The whole genome analysis affords clearer species boundaries over 16S rRNA gene sequencing and traditional phenotypic approaches for the identification and phylogenetic organization of Aerococcus species. The newly described species can be differentiated by matrix-assisted laser desorption ionization time-of-flight analysis of protein signatures. We propose the emendation of the description of A. urinae (type strain ATCC 51268T = CCUG 34223T=NCFB 2893) and the names of Aerococcus tenax sp. nov. (ATCC TSD-302T = DSM 115700T = CCUG 76531T=NR-58630T), Aerococcus mictus sp. nov. (ATCC TSD-301T = DSM 115699T = CCUG 76532T=NR-58629T), and Aerococcus loyolae sp. nov. (ATCC TSD-300T = DSM 115698T = CCUG 76533T=NR-58628T) for three of the newly identified genomic taxa.
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Aerococcus , Aerococcus/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Ácidos Graxos/químicaRESUMO
Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii are dominant species of the urogenital microbiota. Prior studies suggest that these Lactobacillus species play a significant role in the urobiome of healthy females. In our prior genomic analysis of all publicly available L. jensenii and Lactobacillus mulieris genomes at the time (n = 43), we identified genes unique to these two closely related species. This motivated our further exploration here into their genotypic differences as well as into their phenotypic differences. First, we expanded genome sequence representatives of both species to 61 strains, including publicly available strains and nine new strains sequenced here. Genomic analyses conducted include phylogenetics of the core genome as well as biosynthetic gene cluster analysis and metabolic pathway analyses. Urinary strains of both species were assayed for their ability to utilize four simple carbohydrates. We found that L. jensenii strains can efficiently catabolize maltose, trehalose, and glucose, but not ribose, and L. mulieris strains can utilize maltose and glucose, but not trehalose and ribose. Metabolic pathway analysis clearly shows the lack of treB within L. mulieris strains, indicative of its inability to catabolize external sources of trehalose. While genotypic and phenotypic observations provide insight into the differences between these two species, we did not find any association with urinary symptom status. Through this genomic and phenotypic investigation, we identify markers that can be leveraged to clearly distinguish these two species in investigations of the female urogenital microbiota. IMPORTANCE We have expanded upon our prior genomic analysis of L. jensenii and L. mulieris strains, including nine new genome sequences. Our bioinformatic analysis finds that L. jensenii and L. mulieris cannot be distinguished by short-read 16S rRNA gene sequencing alone. Thus, to discriminate between these two species, future studies of the female urogenital microbiome should employ metagenomic sequencing and/or sequence species-specific genes, such as those identified here. Our bioinformatic examination also confirmed our prior observations of differences between the two species related to genes associated with carbohydrate utilization, which we tested here. We found that the transport and utilization of trehalose are key distinguishing traits of L. jensenii, which is further supported by our metabolic pathway analysis. In contrast with other urinary Lactobacillus species, we did not find strong evidence for either species, nor particular genotypes, to be associated with lower urinary tract symptoms (or the lack thereof).
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Maltose , Vagina , Feminino , Humanos , RNA Ribossômico 16S/genética , Lactobacillus/genética , GenótipoRESUMO
Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, e.g., the gut and oral cavity. To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, here we present a bladder-specific bacterial reference collection comprised of 1134 genomes. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2 % of the genera found when we reexamined previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis found that the taxonomies and functions of the bladder microbiota shared more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder E. coli isolates and 387 gut E. coli isolates supports the hypothesis that phylogroup distribution and functions of E. coli strains differ dramatically between these two very different niches. This bladder-specific bacterial reference collection is a unique resource that will enable hypothesis-driven bladder microbiota research and comparison to isolates from other anatomical sites.
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Due to its frequent association with urinary tract infections (UTIs), Escherichia coli is the best characterized constituent of the urinary microbiota (urobiome). However, uropathogenic E. coli is just one member of the urobiome. In addition to bacterial constituents, the urobiome of both healthy and symptomatic individuals is home to a diverse population of bacterial viruses (bacteriophages). A prior investigation found that most bacterial species in the urobiome are lysogens, harboring one or more phages integrated into their genome (prophages). Many of these prophages are temperate phages, capable of entering the lytic cycle and thus lysing their bacterial host. This transition from the lysogenic to lytic life cycle can impact the bacterial diversity of the urobiome. While many phages that infect E. coli (coliphages) have been studied for decades in the laboratory setting, the coliphages within the urobiome have yet to be cataloged. Here, we investigated the diversity of urinary coliphages by first identifying prophages in all publicly available urinary E. coli genomes. We detected 3,038 intact prophage sequences, representative of 1,542 unique phages. These phages include both novel species as well as species also found within the gut microbiota. Ten temperate phages were isolated from urinary E. coli strains included in our analysis, and we assessed their ability to infect and lyse urinary E. coli strains. We also included in these host range assays other urinary coliphages and laboratory coliphages. The temperate phages and other urinary coliphages were successful in lysing urinary E. coli strains. We also observed that coliphages from non-urinary sources were most efficient in killing urinary E. coli strains. The two phages, T2 and N4, were capable of lysing 83.5% (n = 86) of strains isolated from females with UTI symptoms. In conclusion, our study finds a diverse community of coliphages in the urobiome, many of which are predicted to be temperate phages, ten of which were confirmed here. Their ability to infect and lyse urinary E. coli strains suggests that urinary coliphages may play a role in modulating the E. coli strain diversity of the urobiome.
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Bacteriófagos , Microbiota , Feminino , Humanos , Escherichia coli/genética , Colífagos/genética , Bacteriófagos/genética , Lisogenia , Prófagos/genética , BactériasRESUMO
Streptococcus anginosus is a prevalent member of the human flora. While it has been found in the microbiota of "healthy" asymptomatic individuals, it has also been associated with genitourinary tract infections and bacteremia. Based upon multilocus sequence analysis, two subspecies and two genomosubspecies have been characterized for the species. We previously conducted whole-genome sequencing of 85 S. anginosus isolates from the urinary tract. Here, we present genomic analysis of this species, including isolates from the urinary tract as well as gut and fecal, vaginal, oral, respiratory, and blood and heart samples. Average nucleotide identity and core genome analysis revealed that these strains form two distinct groups. Group 1 is comprised of the S. anginosus type strain and other previously identified S. anginosus subspecies and genomosubspecies, including isolates from throughout the human body. In contrast, group 2 consists of predominantly urinary streptococci (n = 77; 85.6%). Both of these S. anginosus groups are distinct from other members of the Streptococcus anginosus group (SAG) species S. intermedius and S. constellatus. Genes conserved among all strains of one group but not in any strains in the other group were next identified. Group 1 strains included genes found in S. intermedius and S. constellatus, suggesting that they were lost within the ancestor of the group 2 strains. In contrast, genes unique to the group 2 strains were homologous to more distant streptococci, indicative of acquisition via horizontal gene transfer. These genes are ideal candidates for use as marker genes to distinguish between the two groups in the human microbiota. IMPORTANCE Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract. In contrast, only 8 urinary strains were identified within the other group, which contained the S. anginosus type strain, as well as all identified subspecies and genomosubspecies. While genomic analysis suggested that this urinary group of S. anginosus is genomically different from the previously characterized S. anginosus subspecies, phenotypic characterization is still needed. Given prior reports of the prevalence of S. anginosus in the urinary tract of both continent and incontinent females, future studies are needed to investigate if the symptom state of the urinary tract is associated with these two different groups.
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Streptococcus anginosus , Infecções Urinárias , Feminino , Humanos , Streptococcus anginosus/genética , Streptococcus/genética , Genômica , Tipagem de Sequências MultilocusRESUMO
Background: Antimicrobial and antifungal species are essential members of the healthy human microbiota. Several different species of lactobacilli that naturally inhabit the human body have been explored for their probiotic capabilities including strains of the species Lactobacillus gasseri. However, L. gasseri (identified by 16S rRNA gene sequencing) has been associated with urogenital symptoms. Recently a new sister taxon of L. gasseri was described: L. paragasseri. L. paragasseri is also posited to have probiotic qualities. Methods: Here, we present a genomic investigation of all (n = 79) publicly available genome assemblies for both species. These strains include isolates from the vaginal tract, gastrointestinal tract, urinary tract, oral cavity, wounds, and lungs. Results: The two species cannot be distinguished from short-read sequencing of the 16S rRNA as the full-length gene sequences differ only by two nucleotides. Based upon average nucleotide identity (ANI), we identified 20 strains deposited as L. gasseri that are in fact representatives of L. paragasseri. Investigation of the genic content of the strains of these two species suggests recent divergence and/or frequent gene exchange between the two species. The genomes frequently harbored intact prophage sequences, including prophages identified in strains of both species. To further explore the antimicrobial potential associated with both species, genome assemblies were examined for biosynthetic gene clusters. Gassericin T and S were identified in 46 of the genome assemblies, with all L. paragasseri strains including one or both bacteriocins. This suggests that the properties once ascribed to L. gasseri may better represent the L. paragasseri species.
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Lactobacillus gasseri , Feminino , Humanos , Lactobacillus gasseri/genética , RNA Ribossômico 16S/genética , Genômica , Boca , Prófagos/genéticaRESUMO
Lactobacilli are dominant members of the healthy female bladder microbiota. Here, we report the draft genome sequences of 4 Lactobacillus jensenii and 3 Lactobacillus mulieris strains isolated from catheterized urine samples.
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Klebsiella variicola, a member of Klebsiella pneumoniae complex, is found to infect plants, insects, and animals and is considered an emerging pathogen in humans. While antibiotic resistance is often prevalent among K. variicola isolates from humans, this has not been thoroughly investigated in isolates from nonhuman sources. Prior evidence suggests that K. variicola can be transmitted between agricultural products as well as between animals, and the use of antibiotics in agriculture has increased antibiotic resistance in other emerging pathogens. Furthermore, in animals that contain K. variicola as a normal member of the rumen microbiota, the same bacteria can also cause infections, such as clinical mastitis in dairy cows. Here, we describe K. variicola UFMG-H9 and UFMG-H10, both isolated from the urine of healthy Gyr heifers. These two genomes represent the first isolates from the urine of cattle and exhibit greater similarity with strains from the human urinary tract than isolates from bovine fecal or milk samples. Unique to the UFMG-H9 genome is the presence of flagellar genes, the first such observation for K. variicola. Neither of the sampled animals had symptoms associated with K. variicola infection, even though genes associated with virulence and antibiotic resistance were identified in both strains. Both strains were resistant to amoxicillin, erythromycin, and vancomycin, and UFMG-H10 is resistant to fosfomycin. The observed resistances emphasize the concern regarding the emergence of this species as a human pathogen given its circulation in healthy livestock animals. IMPORTANCE Klebsiella variicola is an opportunistic pathogen in humans. It also has been associated with bovine mastitis, which can have significant economic effects. While numerous isolates have been sequenced from human infections, only 12 have been sequenced from cattle (fecal and milk samples) to date. Recently, we discovered the presence of K. variicola in the urine of two healthy heifers, the first identification of K. variicola in the bovine urinary tract and the first confirmed K. variicola isolate encoding for flagella-mediated motility. Here, we present the genome sequences and analysis of these isolates. The bovine urinary genomes are more similar to isolates from the human urinary tract than they are to other isolates from cattle, suggesting niche specialization. The presence of antibiotic resistance genes is concerning, as prior studies have found transmission between animals. These findings are important to understand the circulation of K. variicola in healthy livestock animals.
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Infecções por Klebsiella , Animais , Antibacterianos/farmacologia , Bovinos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Humanos , Klebsiella/genética , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/veterinária , Klebsiella pneumoniae/genética , FilogeniaRESUMO
The study of livestock microbiota has immediate benefits for animal health as well as mitigating food contamination and emerging pathogens. While prior research has indicated the gastrointestinal tract of cattle as the source for many zoonoses, including Shiga-toxin producing Escherichia coli and antibiotic resistant bacteria, the bovine urinary tract microbiota has yet to be thoroughly investigated. Here, we describe 5 E. coli and 4 Pseudomonas aeruginosa strains isolated from urine of dairy Gyr cattle. While both species are typically associated with urinary tract infections and mastitis, all of the animals sampled were healthy. The bovine urinary strains were compared to E. coli and P. aeruginosa isolates from other bovine samples as well as human urinary samples. While the bovine urinary E. coli isolates had genomic similarity to isolates from the gastrointestinal tract of cattle and other agricultural animals, the bovine urinary P. aeruginosa strains were most similar to human isolates suggesting niche adaptation rather than host adaptation. Examination of prophages harbored by these bovine isolates revealed similarity with prophages within distantly related E. coli and P. aeruginosa isolates from the human urinary tract. This suggests that related urinary phages may persist and/or be shared between mammals. Future studies of the bovine urinary microbiota are needed to ascertain if E. coli and P. aeruginosa are resident members of this niche and/or possible sources for emerging pathogens in humans.
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Corynebacterium phoceense is a Gram-positive species previously isolated from human urine. Although other species from the same genus have been associated with urinary tract infections, C. phoceense is currently believed to be a non-pathogenic member of the urogenital microbiota. Prior to our study, only two isolates were described in the literature, and very little is known about the species. Here, we describe C. phoceense UFMG-H7, the first strain of this species isolated from the urine of healthy cattle. The genome for this isolate was produced and compared to the two other publicly available C. phoceense as well as other Corynebacterium genome assemblies. Our in-depth genomic analysis identified four additional publicly available genome assemblies that are representatives of the species, also isolated from the human urogenital tract. Although none of the strains have been associated with symptoms or disease, numerous genes associated with virulence factors are encoded. In contrast to related Corynebacterium species and Corynebacterium species from the bovine vaginal tract, all C. phoceense strains examined code for the SpaD-type pili suggesting adherence is essential for its persistence within the urinary tract. As the other C. phoceense strains analysed were isolated from the human urogenital tract, our results suggest that this species may be specific to this niche.
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Corynebacterium/isolamento & purificação , Microbiota , Sistema Urogenital/microbiologia , Animais , Bovinos , Corynebacterium/classificação , Corynebacterium/genética , Genoma Bacteriano , Humanos , Urina/microbiologiaRESUMO
Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Recently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton-Valentine leukocidin (PVL)-associated genes lukF-PV/lukS-PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt.
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Prófagos/isolamento & purificação , Fagos de Staphylococcus/isolamento & purificação , Staphylococcus aureus/virologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Egito , Humanos , Prófagos/classificação , Prófagos/genética , Infecções Estafilocócicas/microbiologia , Fagos de Staphylococcus/classificação , Fagos de Staphylococcus/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , VirulênciaRESUMO
While the gram-positive bacterium Vagococcus fluvialis has been isolated from the environment as well as fish, birds, and mammals, very little is known about the species. V. fluvialis is believed to be a probiotic in fishes. However, within mammals, it is more frequently isolated from infectious tissue, including on rare occasions human and livestock lesions. Prior to the study described here, V. fluvialis had never been found in healthy bovine animals. Here, we present the complete genomes of V. fluvialis UFMG-H6, UFMG-H6B, and UFMG-H7, novel strains isolated from urine samples from healthy bovine females. These are the first genomes of mammalian isolates and the first description of V. fluvialis from urine. The genomes did not encode for any known virulence genes, suggesting that they may be commensal members of the urine microbiota.
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Enterococcaceae , Peixes , Animais , Bovinos , Feminino , VirulênciaRESUMO
Urinary tract infections (UTIs) are one of the most common human bacterial infections. While UTIs are commonly associated with colonization by Escherichia coli, members of this species also have been found within the bladder of individuals with no lower urinary tract symptoms (no LUTS), also known as asymptomatic bacteriuria. Prior studies have found that both uropathogenic E. coli (UPEC) strains and E. coli isolates that are not associated with UTIs encode for virulence factors. Thus, the reason(s) why E. coli sometimes causes UTI-like symptoms remain(s) elusive. In this study, the genomes of 66 E. coli isolates from adult female bladders were sequenced. These isolates were collected from four cohorts, including women: (1) without lower urinary tract symptoms, (2) overactive bladder symptoms, (3) urgency urinary incontinence, and (4) a clinical diagnosis of UTI. Comparative genomic analyses were conducted, including core and accessory genome analyses, virulence and motility gene analyses, and antibiotic resistance prediction and testing. We found that the genomic content of these 66 E. coli isolates does not correspond with the participant's symptom status. We thus looked beyond the E. coli genomes to the composition of the entire urobiome and found that the presence of E. coli alone was not sufficient to distinguish between the urobiomes of individuals with UTI and those with no LUTS. Because E. coli presence, abundance, and genomic content appear to be weak predictors of UTI status, we hypothesize that UTI symptoms associated with detection of E. coli are more likely the result of urobiome composition.
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Lactobacilli are dominant members of the "healthy" female urogenital microbiota. One of these species, Lactobacillus jensenii, is routinely identified in the urinary microbiota of women both with and without urinary tract symptoms. In March 2020, the new bacterial species Lactobacillus mulieris was introduced, and phylogenetic and average nucleotide identity analysis identified eight L. jensenii strains that should be classified as members of the L. mulieris species. This prompted our phylogenomic study of all publicly available L. jensenii and L. mulieris genome sequences. While there is little variation in the 16S rRNA gene sequences, the core genome shows a clear distinction between genomes of the two species. We find eight additional strains of the species L. mulieris among these genomes. Furthermore, one strain, currently classified as L. mulieris UMB7784, is distinct from both L. jensenii and L. mulieris strains. As part of our comparative genomic study, we also investigated the genetic content that distinguishes these two species. Unique to the L. jensenii genomes are several genes related to catabolism of disaccharides. In contrast, L. mulieris genomes encode several cell surface and secreted proteins that are not found within the L. jensenii genomes. These L. jensenii-specific and L. mulieris-specific loci provide insight into phenotypic differences of these two species.IMPORTANCELactobacillus species play a key role in the health of the urinary tract. For instance, Lactobacillus crispatus and L. jensenii have been found to inhibit uropathogenic Escherichia coli growth. While L. crispatus is typically found only within the microbiota of women without lower urinary tract symptoms (LUTS), L. jensenii has been found in the microbiota of women both with and without LUTS. With the recent introduction of the new species Lactobacillus mulieris, several strains of L. jensenii were reclassified as L. mulieris based upon gene marker and average nucleotide identity. We took a phylogenomic and comparative genomic approach to ascertain the genetic determinants of these two species. Looking at a larger data set, we identified additional L. mulieris strains, including one distinct from other members of the species-L. mulieris UMB7784. Furthermore, we identified unique loci in each species that may have clinical implications.
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Genoma Bacteriano , Genômica , Lactobacillus/genética , Filogenia , DNA Bacteriano/genética , Feminino , Humanos , Lactobacillus/classificação , RNA Ribossômico 16S/genética , Especificidade da Espécie , Vagina/microbiologiaRESUMO
In many mammalian species, more than half of the initial oocyte population is eliminated by neonatal life, thus limiting the oocyte reserve for reproduction. The cause or mechanism of this major oocyte loss remains poorly understood. We examined the apoptotic pathway involved in oocyte elimination in wild-type mouse ovaries as well as in Msh5 -/- ovaries, in which all oocytes were eliminated due to a lack of double strand break repair. Immunoblot and immunofluorescence staining showed that an initiator caspase 9 and an effector caspase 7 were constitutively activated in almost all oocytes in fetal ovaries regardless of their genotypes. In caspase 9 -/- ovaries, the total number of oocytes remained high while that in wild-type ovaries steadily declined during ovarian development. Therefore, the activation of caspase 9 was required for but did not immediately lead to oocyte demise. We found that XIAP, an endogenous inhibitor of apoptosis, was also abundant in oocytes during meiotic prophase progression. On the other hand, a cleaved form of PARP1, a target of effector caspases, was localized to the nuclei of a limited number of oocytes, and the frequency of cleaved PARP1-positive oocyte nuclei increased significantly higher before all oocytes disappeared in Msh5 -/- ovaries. We conclude that the mitochondrial apoptotic pathway mediated by caspase 9 is constitutively activated in oocytes and renders the elimination of oocytes with meiotic errors, which can be captured by the cleavage of PARP1.