Pleiotropic Effects of miR5504 Underlying Plant Height, Grain Yield and Quality in Rice.

MicroRNAs (miRNAs) are known to play critical roles in regulating rice agronomic traits through mRNA cleavage or translational repression. Our previous study indicated that miR5504 regulates plant height by affecting cell proliferation and expansion. Here, the two independent homozygous mir5504 mutants (CR1 and CR2) and overexpression lines (OE1 and OE2) were further used to investigate the functions of miR5504. The panicle length, 1000-grain weight and grain yield per plant of miR5504-OE lines were identical to those of Nipponbare (NIP), but the 1000-grain weight of mir5504 mutants was reduced by about 10% and 9%, respectively. Meanwhile, the grain width and thickness of mir5504 mutants decreased significantly by approximately 10% and 11%, respectively. Moreover, the cytological results revealed a significant decrease in cell number along grain width direction and cell width in spikelet in mir5504, compared with those in NIP. In addition, several major storage substances of the rice seeds were measured. Compared to NIP, the amylose content of the mir5504 seeds was noticeably decreased, leading to an increase of nearly 10 mm in gel consistency (GC) in mir5504 lines. Further investigation confirmed that LOC_Os08g16914 was the genuine target of miR5504: LOC_Os08g16914 over-expression plants phenocopied the mir5504 mutants. This study provides insights into the role of miR5504 in rice seed development.

Rice miR5504 regulates plant height by affecting cell proliferation and expansion.

miRNAs play critical roles in the regulation of plant growth and development by cleaving mRNA or repressing transcription. In our previous study, miR5504 with unknown functions was captured by small RNA sequencing. Here, the function and characters of miR5504 were extensively analyzed using CRISPR/Cas9, overexpression strategy, Northern blot, cytological analysis, and transcriptomics analysis. We found that the dwarf phenotype of mir5504 mutants (mir5504-1 and mir5504-2) appeared on 35-day seedlings and became more apparent at the mature stage. The cytological results showed a substantial decrease in the vascular bundle number, cell number and cell length in the mir5504 mutant compared with NIP. In addition, we found that miR5504 regulated plant height by targeting LOC_Os08g16914. The results of RNA-seq revealed that numerous biological processes were mainly enriched, including DNA-binding transcription factor activity, transferase activity, regulation of transcription, metabolic process, and protein binding. Meanwhile, KEEG analysis showed that numerous proteins were associated with cellular processes and metabolism pathways. Taken together, miR5504 may be involved in the regulation of plant height by affecting cell expansion and division of internode in rice.

Inositolphosphorylceramide synthases, OsIPCSs, regulate plant height in rice.

Inositolphosphorylceramide synthase (IPCS) catalyses ceramides and phosphatidylinositol (PI) into inositolphosphorylceramide (IPC), which is involved in the regulation of plant growth and development. A total of three OsIPCS family genes have been identified in rice. However, most of their functions remain unknown. Here, the functions of OsIPCSs were analyzed by CRISPR/Cas9 technology, lipidomics analysis, and transcriptomics analysis. Single-gene mutation of OsIPCSs resulted in dwarf phenotype. Among them, the phenotype of osipcs3 mutant was more severe. Multi-gene mutation of OsIPCS genes led to more severe phenotypes, indicating the additive effects of OsIPCSs. We further determined that a significant decrease in epidermal cell elongation of internode in the mutants. There was a significant decrease in the content of IPC detected in the osipcs2/3 and osipcs1/2/3 mutants. The contents of glycosyl inositol phosphoryl ceramide (GIPC) were also decreased by 20% and 10% in osipcs2/3 and osipcs1/2/3, respectively. The results of RNA-seq showed that numerous DEGs found to be associated with cellular component organization, anatomical structure morphogenesis, and cell growth in the osipcs2, osipcs2/3, and osipcs1/2/3. Taken together, OsIPCSs may be involved in the regulation of plant height through affecting cell growth and sphingolipid metabolism in rice.

Lipid transfer protein, OsLTPL18, is essential for grain weight and seed germination in rice.

Nonspecific lipid transfer proteins (nsLTPs) promote the intermembrane transportation of phospholipids, fatty acids, and steroids, and play diverse roles in various biological processes. However, the potential roles of the rice nsLTPs have not been well elucidated yet. Here, the functions of OsLTPL18 were analyzed using CRISPR/Cas9 strategy and cytological analysis. The osltpl18 (osltpl18-1, osltpl18-2, and osltpl18-3) seeds were thinner, and 1000-grain weight and grain thickness of osltpl18 plants were decreased obviously, compared to the ZH11. Meanwhile, the results of germination assay and 1 % TTC staining showed that vigor of osltpl18 seeds decreased significantly. Furthermore, the results of scanning electron microscopy (SEM) revealed that the cell width of spikelet hull in osltpl18 lines was significantly reduced than that in WT, as well as cell number in grain-width direction. Finally, we found that co-expressed genes were enriched in glucan biosynthesis, protein transporter activity, serine-type endopeptidase inhibitor activity, and nutrient reservoir activity. In this study, we discussed that OsLTPL18 might have coordinating functions in regulation of grain weight and germination in rice.

New Fructokinase, OsFRK3, Regulates Starch Accumulation and Grain Filling in Rice.

Plant fructokinase (FRK) guarantees the growth and development of higher plants by participating in carbohydrate metabolism. In this study, a new fructokinase, OsFRK3, was identified using bioinformatics analysis, enzyme assay, bacterial growth assay, and yeast complementation test. Then, we created OsFRK3 knockout transgenic lines (osfrk3-1 and osfrk3-2) by the CRISPR/Cas9 technology. We found that the 1000-grain weight decreased notably (approximately -3.6% and -6.1%, respectively) in osfrk3-1 and osfrk3-2. Evidently decreased grain width, grain thickness, and endosperm filling rate were detected in the osfrk3 mutants (osfrk3-1 and osfrk3-2) compared with those of the WT. In addition, the content of seed total starch was significantly decreased by 3.42 and 4.80% in osfrk3 lines, compared with that in the WT. The level of maltose was significantly reduced in the mutants, while that of sucrose and fructose was obviously increased in the mutants. The transcript levels of OsGBSS1, OsBEIIb, OsPFP1ß, and OsAGPL1 were significantly decreased in the osfrk3 mutants. These results suggest that OsFRK3 may positively regulate the accumulation of starch through influencing the sugar metabolism.

Glycine- and Proline-Rich Protein OsGPRP3 Regulates Grain Size and Quality in Rice.

The grain size and shape of rice are limited by the growth of the spikelet hulls and are important selective target during domestication and breeding. In this study, we identified a glycine- and proline-rich protein (OsGPRP3), which belongs to a conserved family rarely studied. We found that OsGPRP3 was highly expressed in the seed at 10 days after pollination (DAP) using qRT-PCR, pOsGPRP3::GUS and in situ hybridization. Knockout and knockdown of OsGPRP3 led to significant decrease of 1000-grain weight, grain width, and grain thickness. We further found that the content of storage protein and total lipid were decreased in osgprp3 lines. In particular, the contents of C14:0 (myristic acid), C16:0 (palmitic acid), C18:1 (oleic acid), and C18:2 (linoleic acid) were reduced in osgprp3 lines. Cytological experiments revealed that the cell width of spikelet hull in osgprp3 lines was significantly reduced than that in WT. Taken together, our results reveal that OsGPRP3 regulates the grain size and shape of rice by influencing the cell width of spikelet hulls and the accumulation of storage protein and lipids.

OsDCL3b affects grain yield and quality in rice.

KEY MESSAGE: We reported that knockdown of OsDCL3b decreased grain yield but increased grain quality in rice, which is helpful for molecular breeding in crops. Multiple DICER-LIKE (DCL) genes usually exist and show diverse biochemical and phenotypic functions in land plants. In rice, the biochemical function of OsDCL3b is known to process 24-nucleotide panicle phased small RNAs, however, its phenotypic functions are unclear. Here we reported that knockdown of OsDCL3b led to reduced pollen fertility, seed setting rate, and decreased grain yield but increased grain quality in rice. To reveal the molecular mechanism of the above phenomena, extracted RNAs from rice panicles of the wild type (WT) and OsDCL3b-RNAi line S6-1 were analyzed by deep sequencing. It showed that knockdown of OsDCL3b affected the biogenesis of both 21- and 24-nucleotide small RNAs including miRNAs and phased small RNAs. Using RNA-seq, 644 up- and 530 down-regulated mRNA genes were identified in panicles of line S6-1, and 550 and 273 differentially spliced genes with various alternative splicing (AS) events were observed in panicles of line S6-1 and WT, respectively, suggesting that OsDCL3b involved in influencing the transcript levels of mRNA genes and the AS events in rice panicles. Thus, our results show that knockdown of OsDCL3b will affect the biogenesis of small RNAs, which is involved in regulating the transcription of mRNA genes, and consequently influence the grain yield and quality in rice.

Expression and function analysis of a rice OsHSP40 gene under salt stress.

Heat shock proteins (HSPs) play essential roles in both plant growth and abiotic stress tolerance. In rice, OsHSP40 was recently reported to regulate programmed cell death (PCD) of suspension cells under high temperature. However, the expression and functions of OsHSP40 under normal growth or other abiotic stress conditions is still unknown. We reported the expression and function of a rice OsHSP40 gene under salt stress. Homologous proteins of OsHSP40 were collected from the NCBI database and constructed the neighbor-joining (NJ) phylogenetic tree. The expression pattern of OsHSP40 was detected by qRT-PCR under NaCl (150 mM) treatment. Then, identified a rice T-DNA insertion mutant oshsp40. At last, we compared and analyzed the phenotypes of oshsp40 and wild type under salt stress. OsHSP40 was a constitutively expressed small HSP (sHSP) gene and was close related to other plant sHSPs. Moreover, the expression of OsHSP40 was regulated by salt, varying across time points and tissues. Furthermore, the growth of T-DNA insertion mutant of OsHSP40 (designated as oshsp40) was suppressed by NaCl (150 mM) compared with that of the WT at seedling stage. Detailed measurement showed root and shoot length of the oshsp40 seedlings were significantly shorter than those of the WT seedlings under NaCl stress. In addition, the pot experiment results revealed that seedlings of oshsp40 withered more seriously compared with those of WT after NaCl treatment and recovery, and that survival rate and fresh weight of oshsp40 seedlings were significantly reduced. Taken together, these data suggested that OsHSP40 had multiple functions in rice normal growth and abiotic stress tolerance.

Towards a global phylogeny of freshwater mussels (Bivalvia: Unionida): Species delimitation of Chinese taxa, mitochondrial phylogenomics, and diversification patterns.

The Yangtze River Basin in China is one of the global hotspots of freshwater mussel (order Unionida) diversity with 68 nominal species. Few studies have tested the validity of these nominal species. Some taxa from the Yangtze unionid fauna have not been adequately examined using molecular data and well-positioned phylogenetically with respect to the global Unionida. We evaluated species boundaries of Chinese freshwater mussels, and disentangled their phylogenetic relationships within the context of the global freshwater mussels based on the multi-locus data and complete mitochondrial genomes. Moreover, we produced the time-calibrated phylogeny of Unionida and explored patterns of diversification. COI barcode data suggested the existence of 41 phylogenetic distinct species from our sampled 40 nominal taxa inhabiting the middle and lower reaches of the Yangtze River. Maximum likelihood and Bayesian inference analyses on three loci (COI, 16S, and 28S) and complete mitochondrial genomes showed that the subfamily Unioninae sensu stricto was paraphyletic, and the subfamily Anodontinae should be subsumed under Unioninae. In addition, we described two new tribes (Aculamprotulini tribe nov. and Lepidodesmini tribe nov.) in the subfamily Unioninae and one new genus (Parvasolenaiagen. nov.) in the subfamily Gonideinae. Molecular dating analysis suggested freshwater mussels diversified at 346.1 Mya (HPD = 286.6-409.9). The global diversification rate for Unionida was estimated to be 0.025 species/Myr. Our study found only a single well-supported rate shift in Unionida diversification, occurring at the base of the subfamily Ambleminae. The evolution of active host-attraction may have triggered the burst of speciation in Ambleminae, and the environment and geography of the Mississippi River Basin likely sustained this radiation.

Gestational exposure to titanium dioxide nanoparticles impairs the placentation through dysregulation of vascularization, proliferation and apoptosis in mice.

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) have recently found applications in a wide variety of consumer goods. TiO2 NPs exposure significantly increases fetal deformities and mortality. However, the potential toxicity of TiO2 NPs on the growth and development of placenta has been rarely studied during mice pregnancy. PURPOSE: The objective of this study was to investigate the effects of maternal exposure of TiO2 NPs on the placentation. METHODS: Mice were administered TiO2 NPs by gavage at 0, 1 and 10 mg/kg/day from gestational day (GD) 1 to GD 13. Uteri and placentas from these mice were collected and counted the numbers of implanted and resorbed embryo and measured the placental weight on GD 13. Placental morphometry was observed by hematoxylin and eosin staining. The levels of Hand1, Esx1, Eomes, Hand2, Ascl2 and Fra1 mRNA were assessed by qRT-PCR. Uterine NK (uNK) cells were detected by using DBA lectin. Laminin immunohistochemical staining was to identify fetal vessels. Western blotting and transmission electron micrograph (TEM) were used to assess the apoptosis of placenta. RESULTS: No treatment-related difference was observed in the numbers of implanted and resorbed embryos and weight of placenta between the groups. However, 1 mg/kg/day TiO2 NPs treatment significantly reduced the ratio of placenta/body weight on GD 13. The proportion of spongiotrophoblast in the 10 mg/kg/day dose group became higher than that in the control group, yet that of labyrinth was significantly lower in 10 mg/kg/day mice. The expression levels of Hand1, Esx1, Eomes, Hand2, Ascl2 and Fra1 mRNA markedly decreased in TiO2 NP treated placentas. Furthermore, TiO2 NPs treatment impaired the formation of intricate networks of fetal vessels and reduced the number of uNK cells, and inhibited proliferation and induced apoptosis of placenta by nuclear pyknosis, the activation of caspase-3 and upregulation of Bax protein and downregulation of Bcl-2 protein on GD 13. CONCLUSION: Gestational exposure to TiO2 NPs significantly impairs the growth and development of placenta in mice, with a mechanism that seems to be involved in the dysregulation of vascularization, proliferation and apoptosis. Therefore, our results suggested the need for great caution while handling of the nanomaterials by workers and specially pregnant consumers.

Down-Regulation of Neuropathy Target Esterase in Preeclampsia Placenta Inhibits Human Trophoblast Cell Invasion via Modulating MMP-9 Levels.

BACKGROUND/AIMS: Neuropathy target esterase (NTE, also known as neurotoxic esterase) is proven to deacylate phosphatidylcholine (PC) to glycerophosphocholine as a phospholipase B. Recently; studies showed that artificial phosphatidylserine/PC microvesicles can induce preeclampsia (PE)-like changes in pregnant mice. However, it is unclear whether NTE plays a key role in the pathology of PE, a pregnancy-related disease, which was characterized by deficient trophoblast invasion and reduced trophoblast-mediated remodeling of spiral arteries. The aim of this study was to investigate the expression pattern of NTE in the placenta from women with PE and normal pregnancy, and the molecular mechanism of NTE involved in the development of PE. METHODS: NTE expression levels in placentas from 20 pregnant women with PE and 20 healthy pregnant women were detected using quantitative PCR and immunohistochemistry staining. The effect of NTE on trophoblast migration and invasion and the underlying mechanisms were examined in HTR-8/SVneo cell lines by transfection method. RESULTS: NTE mRNA and protein expression levels were significantly decreased in preeclamptic placentas than normal control. Over-expression of NTE in HTR-8/SVneo cells significantly promoted trophoblast cells migration and invasion and was associated with increased MMP-9 levels. Conversely, shRNA-mediated down-regulation of NTE markedly inhibited the cell migration and invasion. In addition, silencing NTE reduced the MMP-9 activity and phosphorylated Erk1/2 and AKT levels. CONCLUSIONS: Our results suggest that the decreased NTE may contribute to the development of PE through impairing trophoblast invasion by down-regulating MMP-9 via the Erk1/2 and AKT signaling pathway.

Cross-protection of Lactococcus lactis-displayed HA2 subunit against homologous and heterologous influenza A viruses in mice.

Current influenza vaccines provide strain-specific protection against homologous subtypes and need to be updated annually. Therefore, it is essential to develop a universal vaccine that would induce broadly cross-protective immunity against homologous and heterologous influenza A viruses. The highly conserved HA2 subunit is a promising candidate for developing a universal influenza vaccine. Here, we hypothesized that the HA2 subunit could be displayed on the surface of Lactococcus lactis (L. lactis), using Spax as an anchor protein (L. lactis/pNZ8008-Spax-HA2) and that L. lactis/pNZ8008-Spax-HA2 would have immunogenicity by oral administration without the use of adjuvant in the mouse model. To address this hypothesis, we show that oral vaccination of mice with L. lactis/pNZ8008-Spax-HA2 elicited significant humoral and mucosal immune responses. Importantly, L. lactis/pNZ8008-Spax-HA2 provided 100% protection against homologous H5N1 or heterologous H1N1 virus challenge. These results suggest that an HA2 subunit presented on the surface of L. lactis is an effective universal vaccine candidate against influenza A viruses in the poultry industry and in humans.

Broadly protective immunity against divergent influenza viruses by oral co-administration of Lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin B subunit in mice.

BACKGROUND: Current influenza vaccines need to be annually reformulated to well match the predicated circulating strains. Thus, it is critical for developing a novel universal influenza vaccine that would be able to confer cross-protection against constantly emerging divergent influenza virus strains. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Influenza virus nucleoprotein (NP) is a structural protein which encapsidates the negative strand viral RNA, and anti-NP antibodies play role in cross-protective immunity. Lactococcus lactis (L. lactis) is an ideal vaccine delivery vehicle via oral administration route. However, L. lactis vectored vaccine exhibits poor immunogenicity without the use of mucosal adjuvant. To enhance the immunogenicity of L. lactis vectored vaccine, cholera toxin B (CTB) subunit, one of mucosal adjuvants, is a safe adjuvant for oral route, when combined with L. lactis vectored vaccine. In this study, we hypothesized that pNZ8008, a L. lactis expression plasmid, encoding NP antigen, would be able to elicit cross-protection with the use of CTB via oral administration route. RESULTS: To construct L. lactis vectored vaccine, nucleoprotein (NP) gene of A/California/04/2009(H1N1) was sub-cloned into a L. lactis expression plasmid, pNZ8008. The expression of recombinant L. lactis/pNZ8008-NP was confirmed by Western blot, immunofluorescence assay and flow cytometric analysis. Further, immunogenicity of L. lactis/pNZ8008-NP alone or adjuvanted with cholera toxin B (CTB) subunit was evaluated in a mouse model via oral administration route. Antibodies responses were detected by ELISA. The result indicated that oral administration of L. lactis/pNZ8008-NP adjuvanted with CTB could elicit significant humoral and mucosal immune responses, as well as cellular immune response, compared with L. lactis/pNZ8008-NP alone. To further assess the cross-protective immunity of L. lactis/pNZ8008-NP adjuvanted with CTB, we used L. lactis/pNZ8110-pgsA-HA1 alone or adjuvanted with CTB as controls. Mice that received L. lactis/pNZ8008-NP adjuvanted with CTB were completely protected from homologous H1N1 virus and showed 80% protection against heterologous H3N2 or H5N1 virus, respectively. By contrast, L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB also conferred 100% protection against H5N1 virus infection, but indicated no cross-protection against H1N1 or H5N1 virus challenge. As controls, mice vaccinated orally with L. lactis/pNZ8008-NP alone or L. lactis/pNZ8110-pgsA-HA1 alone could not survive. CONCLUSION: This study is the first to report the construction of recombinant L. lactis/pNZ8008-NP and investigate its immunogenicity with the use of CTB. Compared with L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB, our data support 5 × 10(11) CFU of L. lactis/pNZ8008-NP adjuvanted with 1 µg of CTB is a better combination for universal influenza vaccines development that would provide cross-protective immunity against divergent influenza A viruses.

Protective immunity against influenza H5N1 virus challenge in chickens by oral administration of recombinant Lactococcus lactis expressing neuraminidase.

BACKGROUND: Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat in poultry. Current influenza vaccines predominantly focus on hemagglutinin (HA) which anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. However, Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. The aim of this study was to evaluate the protective immunity of NA based on Lactococcus lactis (L.lactis) expression system against homologous H5N1 virus challenge in a chicken model. RESULTS: L.lactis/pNZ2103-NA which NA is derived from A/Vietnam/1203/2004 (H5N1) (VN/1203/04) was constructed based on L.lactis constitutive expression system in this study. Chickens vaccinated orally with 10(12) colony-forming unit (CFU) of L.lactis/pNZ2103-NA could elicit significant NA-specific serum IgG and mucosa IgA antibodies, as well as neuraminidase inhibition (NI) titer compared with chickens administered orally with saline or L.lactis/pNZ2103 control. Most importantly, the results revealed that chickens administered orally with L.lactis/pNZ2103-NA were completely protected from a lethal H5N1 virus challenge. CONCLUSIONS: The data obtained in the present study indicate that recombinant L.lactis/pNZ2103-NA in the absence of adjuvant can be considered an effective mucosal vaccine against H5N1 infection in chickens via oral administration. Further, these findings support that recombinant L.lactis/pNZ2103-NA can be used to perform mass vaccination in poultry during A/H5N1 pandemic.

Intranasal immunization of recombinant Lactococcus lactis induces protection against H5N1 virus in ferrets.

The increasing outbreaks of highly pathogenic avian influenza A (HPAI) H5N1 viruses in birds and human bring out an urgent need to develop a safe and effective vaccine to control and prevent H5N1 infection. Lactococcus lactis (L. lactis) based vaccine platform is a promising approach for mucosal H5N1 vaccine development. Intranasal immunization is the potential to induce mucosal immune response which is associated with protective immunity. To develop a safe and effective mucosal vaccine against HAPI H5N1, we extended our previous study by evaluating the immunogenicity of L. lactis-psA-HA1 in the absence of adjuvant via intranasal route in the ferret model. Ferrets administered intranasally with L. lactis-pgsA-HA1 could elicit robust humoral and mucosal immune responses, as well as significant HI titers. Importantly, ferrets were completely protected from H5N1 virus challenge. These findings suggest that L. lactis-pgsA-HA1 can be considered an alternative mucosal vaccine during A/H5N1 pandemic.

Lactococcus lactis displayed neuraminidase confers cross protective immunity against influenza A viruses in mice.

Influenza A viruses pose a serious threat to public health. Current influenza A vaccines predominantly focus on hemagglutinin (HA) and show strain-specific protection. Neuraminidase (NA) is much less studied in the context of humoral immunity against influenza A viruses. The purpose of this study is to evaluate the cross protective immunity of NA presented on Lactococcus lactis (L.lactis) surface against homologous and heterologous influenza A viruses in the mouse model. L.lactis/pNZ8110-pgsA-NA was constructed in which pgsA was used as an anchor protein. Mice vaccinated orally with L.lactis/pNZ8110-pgsA-NA could elicit significant NA-specific serum IgG and mucosa IgA antibodies, as well as neuraminidase inhibition (NI) titers. Importantly, L.lactis/pNZ8110-pgsA-NA provided 80% protection against H5N1, 60% protection against H3N2 and H1N1, respectively. These findings suggest that recombinant L.lactis/pNZ110-pgsA-NA in the absence of adjuvant via oral administration can be served as an effective vaccine candidate against diverse strains of influenza A viruses.

RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.