Preimplantation embryology: The molecular genetics of the zona pellucida: mouse mutations and infertility

The zona pellucida is an extracellular matrix surrounding growingoocytes, ovulated eggs and the preimplantation embryo. Aftermediating the relatively species-specific events of fertilization,the zona pellucida provides a post-fertilization block to polyspermyand protects the growing embryo as it passes down the oviduct.The genes that encode the three zona pellucida proteins (ZP1,ZP2, ZP3) have been characterized in mouse and human. The abilityto genetically manipulate the zona pellucida genes in mousemodels has enhanced our knowledge of zona pellucida structureand function in vivo and may translate into a better understandingof human fertility. fertilization/infertility/transgenic mice/zona pellucida/ZP3 null mutation     

Antibodies to human ZP3 induce reversible contraception in transgenic mice with 'humanized' zonae pellucidae

The initial spermatozoon-egg interaction of mammalian fertilization is mediated by the zona pellucida, an extracellular matrix composed of three glycoproteins (ZP1, ZP2, ZP3). These proteins are sufficiently conserved between human and mouse to form chimeric zonae pellucidae, and genetically engineered mice in which the endogenous mouse ZP3 has been replaced by human ZP3 have 'humanized' zonae, but normal fertility. Administration of monoclonal antibodies to mouse ZP3 does not affect fertility in these animals, but administration of antibodies to human ZP3 results in long-term, reversible contraception. The antibodies coat the zonae pellucidae surrounding growing oocytes within the ovary and their presence in the zona matrix inhibits, but does not eliminate, sperm binding. The contraceptive effect is attributed to steric hindrance that decreases sperm binding and prevents penetration through the zona pellucida. The resumption of fertility is associated with the disappearance of antibodies from the zona matrix. No adverse effect on mating behaviour, ovarian histology or fetal development (if administered after fertilization) is detected in treated females. These results suggest that transgenic mice expressing human proteins will prove useful in assessing contraceptive efficacy of zona epitopes in the rational design of immunocontraception directed at the human zona pellucida.     

Structure and function of the proteins of the mammalian Zona pellucida

The zona pellucida (ZP) is the extracellular matrix that plays important roles in sperm-egg interaction. The ZP is composed of three major glycoproteins that exhibit heterogeneity due to extensive post-translational modifications including glycosylation and sulfation. Because of these modifications the nomenclature of ZP proteins from different species based on electrophoretic mobilities has been confusing. As the cDNAs and genes encoding the different ZP proteins have been isolated and sequenced, it is now possible to relate these ZP proteins according to gene families. Using the mouse ZP nomenclature, the ZP proteins from different mammalian species can be classified into three protein families: ZP1, ZP2, and ZP3. Although some of the structural domains of the ZP proteins of different species are conserved within each family, they exhibit distinct biological properties. In the mouse it has been established that ZP3 is the primary sperm receptor while ZP2 has secondary sperm receptor properties. In the pig, however, ZP1 has been shown to have sperm receptor activity similar to that observed in the rabbit and nonhuman primates. It is of interest that the human ZP2 and ZP3 gene families are 60-70% conserved with respect to the mouse ZP amino acid sequence, while the mouse ZP1 is only 39% conserved with respect to human ZP1. Such differences in protein structure and glysosylation may explain the marked species differences in the biochemical, physicochemical and immunochemical properties of the ZP. Studies have now shown that the proteins of the ZP are expressed in a stage specific manner and that there is increasing evidence that ZP proteins are expressed by both granulosa cells and the oocyte and may play a role in granulosa cell differentiation.     

Contraceptive vaccines based on the zona pellucida glycoproteins for dogs and other wildlife population management

Zona pellucida (ZP) glycoproteins, by virtue of their critical role in fertilization, have been proposed as candidate antigens for the development of contraceptive vaccines. In this review, the potential of a ZP-based contraceptive vaccine for the management of wildlife population, with special reference to street dogs, is discussed. Immunization of various animal species, including female dogs, with native porcine ZP led to inhibition of fertility, which was associated with the ovarian dysfunction. Immunization of female dogs with Escherichia coli-expressed recombinant dog ZP glycoprotein-3 (ZP3) either coupled to diphtheria toxoid or expressed as fusion protein with 'promiscuous' T non-B-cell epitope of tetanus toxoid also led to inhibition of fertility. To improve the contraceptive efficacy of ZP-based contraceptive vaccine, various groups are working on improving the immunogen, use of DNA vaccine as prime-boost strategy, and delivering the zona proteins/peptides presented on either virus-like particles or entrapped in microsphere. Host-specific live vectors such as ectromelia virus and cytomegalovirus have also been used to deliver mouse ZP3 in mice. Various studies show the enormous potential of the ZP-based vaccine for the management of wildlife population, where permanent sterilization may be desirable.     

The testis-specifically expressed gene Trim69 is not essential for fertility in mice

Protein ubiquitination is essential for diverse cellular functions including spermatogenesis.The tripartite motif(TRIM)family proteins,most of which have E3 ubiquitin ligase activity,are highly conserved in mammals.They are involved in important cellular processes such as embryonic development,immunity,and fertility.Our previous studies indicated that Trim69,a testis-specific expressed TRIM family gene,potentially participates in the spermatogenesis by mediating testicular cells apoptosis.In this study,we investigated the biological functions of Trim69 in male mice by established Trim69 knockout mice with CRISPR/Cas9 genomic editing technology.Here,we reported that the male Trim69 knockout mice had normal fertility.The adult knockout mice have shown that the appearance of testes,testis/body weight ratios,testicular histomorphology,and the number and quality of sperm were consistent with wild-type mice.These results indicated that the E3 ubiquitin ligase protein Trim69 was not essential for male mouse fertility,and it might be compensated by other TRIM family members such as Trim58 in Trim69-deficiency testis.This study would help to elucidate the functions of tripartite motif protein family and the regulation of spermatogenesis.     

Antifertility Characteristics of the N‐terminal Region of Mouse Equatorial Segment Protein

To investigate antifertility characteristics of the equatorial segment protein (ESP) and its potential immunocontraceptive effect, three partially overlapping cDNA fragments P1/P2/P3, together covering the entire mouse ESP, were cloned, expressed, and purified. The roles of P1/P2/P3 in fertility were investigated through in vitro fertilization and mouse mating test. Antibodies against P1/P2 significantly reduced the rates of fertilization in vitro in the zona‐intact experiments. Coincubation of zona‐free mouse oocytes with capacitated mouse spermatozoa in the presence of antibodies against P1/P2 also inhibited sperm‐oolemma binding and fusion, while anti‐P3 antibody virtually had no effect on in vitro fertilization at the same concentration. Immunization of female BALB/c mice with N‐terminal of mouse ESP (recombinant P1 and P2) resulted in a significant decrease in the fertility rate as well as the litter size. Double immunofluorescence staining showed that mouse ESP protein was localized to the equatorial segment of acrosome of mouse sperm, and was exposed and surface‐accessible after acrosome reaction. Mouse ESP was also demonstrated to have complementary binding sites on the mouse egg plasma membrane by indirect immunofluorescence assay. These findings suggest that the N‐terminal of mouse ESP could play an important role in fertility and might be a vaccine candidate for contraception. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of human zona pellucida glycoproteins

The human egg may only be fertilized by one spermatozoon toprevent polyploidy. In most mammals, the primary block to polyspermyoccurs at the zona pellucida (ZP). Little is known of the humanZP and the changes occurring following fertilization to preventpolyploidy. Using antibodies directed against synthetic peptidespredicted from the human ZP2 and ZP3 cDNA, we identified ZP3as a 53–60 kDa glycoprotein and ZP2 as a 90–110 kDaglycoprotein in prophase-I oocytes. Characterization of theZP from metaphase II arrested eggs (inseminated–unfertilizedand fertilized–uncleaved), shows no visible modificationof ZP3, but demonstrates that ZP2 undergoes limited proteolysisin the amino terminal domain, to a 60–73 kDa species, denotedZP2_p, which remains linked to the proteolysed fragments by intramoleculardisulphide bonds. A lack of ZP2 proteolytic activity in acrosomalsupernatants is consistent with an oocyte origin for the protease.The ZP2-specific protease may be released during cortical granuleexocytosis which occurs during meiotic maturation and followingsperm–egg fusion as part of the block to polyspermy. Sincemouse ZP2 acts as a secondary sperm receptor, it is possiblethat intact ZP2 binds a secondary egg binding protein, whereascleaved ZP2 does not, suggesting a possible mechanism for theblock to polyspermy. glycosylation/human zona pellucida/proteolysis/ZP2/ZP3 ⁴ Current address: Centre for Immunology, St Vincent's Hospitaland University of New South Wales, Sydney, 2010, Australia ⁵ To whom correspondence should be addressed     

ZP2 pathogenic variants cause in vitro fertilization failure and female infertility

PurposeThe oocyte-borne genetic causes leading to fertilization failure are largely unknown. We aimed to identify novel human pathogenic variants (PV) and genes causing fertilization failure.MethodsWe performed exome sequencing for a consanguineous family with a recessive inheritance pattern of female infertility characterized by oocytes with a thin zona pellucida (ZP) and fertilization failure in routine in vitro fertilization. Subsequent PV screening of ZP2 was performed in additional eight unrelated infertile women whose oocytes exhibited abnormal ZP and similar fertilization failure. Expression of ZP proteins was assessed in mutant oocytes by immunostaining, and functional studies of the wild-type and mutant proteins were carried out in CHO-K1 cells.ResultsTwo homozygous s PV (c.1695-2A>G, and c.1691_1694dup (p.C566Wfs*5), respectively) of ZP2 were identified in the affected women from two unrelated consanguineous families. All oocytes carrying PV were surrounded by a thin ZP that was defective for sperm-binding. Immunostaining indicated a lack of ZP2 protein in the thin ZP. Studies in CHO cells showed that both PV resulted in a truncated ZP2 protein, which might be intracellularly sequestered and prematurely interacted with other ZP proteins.ConclusionWe identified loss-of-function PV of ZP2 causing a structurally abnormal and dysfunctional ZP, resulting in fertilization failure and female infertility.     

Localization of ubiquitin C-terminal hydrolase L1 in mouse ova and its function in the plasma membrane to block polyspermy

Protein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes.     

Characterization of mouse embryonic fibroblasts derived from Rassf6 knockout mice shows the implication of Rassf6 in the regulation of NF-κB signaling

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. We have reported using human cancer cell lines that RASSF6 induces apoptosis and cell cycle arrest via p53 and plays tumor suppressive roles. In this study, we generated Rassf6 knockout mice by CRISPR/Cas technology. Contrary to our expectation, Rassf6 knockout mice were apparently healthy. However, Rassf6-null mouse embryonic fibroblasts (MEF) were resistant against ultraviolet (UV)-induced apoptosis/cell cycle arrest and senescence. UV-induced p53-target gene expression was compromised, and DNA repair was delayed in Rassf6-null MEF. More importantly, KRAS active mutant promoted the colony formation of Rassf6-null MEF but not the wild-type MEF. RNA sequencing analysis showed that NF-κB signaling was enhanced in Rassf6-null MEF. Consistently, 7,12-dimethylbenz(a)anthracene (DMBA) induced skin inflammation in Rassf6 knockout mice more remarkably than in the wild-type mice. Hence, Rassf6 deficiency not only compromises p53 function but also enhances NF-κB signaling to lead to oncogenesis.     

The distribution of neuron‐specific gene family member 1 in brain and germ cells: Implications for the regulation of germ‐line development by brain

Vesicular acidification at early endosomes dissociates endocytosed receptor‐ligand complexes. The ligands, receptors, or both are then directed to late endosomes for degradation or recycled back to the plasma membrane. Of neuron‐specific gene (NSG) family members, early endosomal protein neuron‐specific gene family member 1 (NSG1) is the most important in receptor recycling. In this study, we characterized chicken NSG1 (cNSG1). We found several functional sites related to endocytotic machinery in cNSG1 that were highly conserved with most other vertebrate NSG1 proteins. We examined the tissue and duration specificity and the temporal and spatial patterns of cNSG1 expression. cNSG1 expression was preferentially located in all regions of the brain, neuroendocrine glands, and spinal cord. Unexpectedly, cNSG1 expression was strongly detected during male and female germ‐line development. Expression of NSG1 in two apparently unrelated cell types such as neurons and germ cells suggests NSG1 roles in neurons and germ‐cells chemotaxis and endocytotic machinery. Developmental Dynamics 240:850–861, 2011. © 2011 Wiley‐Liss, Inc.     

Compound heterozygous ZP1 mutations cause empty follicle syndrome in infertile sisters

Empty follicle syndrome (EFS) is a condition in which no oocyte is retrieved from mature follicles after proper ovarian stimulation in an in vitro fertilization procedure. Genetic evidence accumulates for the etiology of recurrent EFS without pharmacological or iatrogenic problems. In this study, we present two infertile sisters in a family with EFS after three cycles of standard ovarian stimulation with human chorionic gonadotrophin and/or gonadotropin‐releasing hormone agonist therapy. Via whole‐exome sequencing and cosegregation test, we identified compound heterozygous mutations in the gene of ZP1 in both of the infertile sisters. Coimmunoprecipitation tests and homology modeling analysis confirmed that both mutated ZP1 disrupt the formation of oocyte zona pellucida by interrupting the interaction among ZP1, ZP2, and ZP3. We thus propose that the specific mutations in ZP1 gene render a causality for the intractable EFS.     

Proacrosin-deficient mice and zona pellucida modifications in an experimental model of multifactorial infertility

In humans, male and female partners contribute more or less equally to the infertility problem. In approximately 20% of infertile couples, the concurrence of male and female factors is suggested to be responsible for infertility. Neither of these factors are known nor is there a model system to prove this assumption. We present such a model system in the mouse, in which the lack of acrosin in the male and modifications of the zona pellucida (ZP) in the female result in a significant reduction of the fertilization rate in vitro. We generated mice carrying a deletion in the proline-rich region (PRR) of the proacrosin gene, resulting in the absence of proacrosin in the homozygous PRR(-/-) male mouse. Under normal conditions, sperm from the proacrosin-deficient mice are still capable of ZP penetration and fertilization. In this study, modifications of the ZP of oocytes after superovulation were achieved by treatment with dimethylsulphoxide or aroclor-1254 or by in-vitro ageing. It is known that under these conditions, a time-dependent hardening of the ZP occurs. The rates of fertilization in vitro of treated and aged oocytes using sperm from PRR(-/-) mice were found to be significantly reduced when compared with those reached with wild-type sperm. The relevance of the acrosin status and ZP condition for fertilization success were further substantiated by the finding that the fertilization rate with PRR(-/-) sperm is affected by the thickness of the ZP. Our results demonstrate that the lack of acrosin in sperm in combination with modifications to the ZP can affect fertility and can be an experimental model for the study of unexplained infertility in human couples in which both male- and female-derived factors are suggested to be the underlying causes.     

Influence of the genetic pattern and sex of mice in experimental paracoccidioidomycosis

Eight genetically different strains of mice were compared regarding the dissemination of Paracoccidioides brasiliensis to the lungs, liver and omentum/pancreas, DTH responses and specific antibody production at 16 weeks after intraperitoneal infection with Pb18, a virulent P. brasiliensis isolate. The degree of dissemination of the infection varied: B10.A and C57Bl/6, the most susceptible mouse strains, had positive cultures and high colony-forming unit (CFU) counts in all analysed organs. DBA/2 and A/Sn mice had negative cultures, being thus classified as the most resistant strains. CBA/J, C3H/HeJ, F₁(A/SnxB10.A) and BALB/c mice were regarded as relatively resistant, since discrete fungal growth was observed only in one or two of the studied organs. All mouse strains, except B10.A mice, produced specific DTH responses which did not seem to be associated with the severity of disease. Production of high levels of specific antibodies was found in all strains except in the DBA/2 and C57B1/6 mice. The influence of the host sex on the outcome of paracoccidioidomycosis was evident only in susceptible animals: female B10.A mice displayed lower CFU counts in the three examined organs, whereas no differences were found between male and female A Sn animals. The higher resistance of female B10.A mice was not accompanied by differences in their capacity to maintain a DTH reaction, nor in their production of antibody. This fact argues against the widely believed association of susceptibility to P. brasiliensis infection with both impaired DTH reactivity and increased humoral response.     

Molecular aspects of mammalian fertilization

The initial communication between the gametes is a molecular,receptor-mediated process that takes place at the surface ofthe egg coat. The zona pellucida plays a central role in thisprocess such that, on the one hand, spermatozoa may bind toit and, on the other hand, it prevents polyspermy. In the mouse,ZP3, a glycoprotein of the zona pellucida with a mol. wt of84 kd, serves as a sperm receptor. Only a relatively small partof ZP3, namely certain O-linked carbohydrate side chains, isinvolved in the process of binding. These oligosaccharides probablybecome bound to enzymes associated with the plasma membraneof the sperm head and thus form an enzyme-substrate complex.A terminal -galactose has been found to be one of the decisivesugar molecules and, moreover, the critical chemical group.After sperm binding to the zona pellucida has taken place, thepolypeptide chain of ZP3 initiates the acrosome reaction inthe sperm head. In the mouse, numerous binding proteins havebeen detected in the sperm plasma membrane: these are enzymessuch as glycosyl transferases, proteinases, and glycosidases.A galactosyl transferase has been found on the surface of themouse sperm that binds specifically to N-acetylglucosamine inthe mouse zona pellucida. It is therefore apparent that carbohydrate-bindingproteins on the sperm surface mediate gamete recognition throughtheir high affinity and specificity for complex glycoconjugatesin the egg coat. In fact, it is not at all surprising that complementarycell-surface protein and glycoconjugates are involved in fertilization,since many somatic cells exhibit a similar mechanism of cellrecognition.     

B lymphocyte recognition of cytochrome c: higher frequency of cells specific for self versus foreign antigen early in the immune response and V gene usage in the response to self antigen

To study immunoglobulin gene usage in the antibody response of mice to the self antigen (Ag) mouse cytochrome c (cyt), B cell hybridomas were prepared from splenic B cells of immunized BALB/c mice prior to the onset of somatic mutation, i.e. 3 days after injecting ovalbumin (OVA)-primed mice with mouse cyt coupled to OVA. Monoclonal antibodies (mAb) from all of the seven primary hybridomas we obtained were sensitive to a single amino acid substitution from aspartic acid to glutamic acid at position 62 in mouse cyt. This is the specificity of the vast majority of B cells responding to mouse cyt as determined from assays of B cells activated in splenic fragment cultures. Six of the mAb derive from the 19.1.2 J558 V_H gene which is also used in the response to α (1 → 6) dextran and three of these mAb derive from the R9 V_x gene, a member of the V_xOx-1 family. The other mAb derive from distinct, although similar, V_x genes. Attempts to obtain hybridomas secreting primary (unmutated) mAb specific for cyt foreign to mice have been hampered by the much lower frequency of B cells responding early to foreign cyt in comparison to the self Ag. This suggests that, contrary to expectation of tolerance mechanisms, in naive BALB/c mice B lymphocytes specific for a single epitope on self cyt are present in higher frequency than B lymphocytes specific for similar epitopes on foreign cyt. Possible explanations for this result include biased expression in the B cell repertoire of the particular combination of V genes encoding mouse cyt-specific mAb or to positive selection of developing B lymphocytes by endogenous Ag.     

实验性自身免疫性卵巢炎卵巢功能早衰的机理研究

目的：探讨卵透明带免疫对卵巢功能的损伤及作用机理。方法：分别以猪卵透明带抗原和缓冲液通过皮内及两后脚掌多点注射免疫Ｂａｌｂ／ｃ小鼠，每２ｗ免疫１次，连续免疫３次。经３次免疫后，测定外周血雌二醇浓度和血清抗卵透明带抗体滴度，观察卵巢组织病理形态学及免疫组化，分析ＩＬ－１，ＥＧＦ在卵巢中的表达。结果：免疫小鼠外周血抗ＳＰ抗体滴度明显高于对照组，而雌二醇浓度则明显低于对照组，且二者存在显著负相关。     

Fertility defects in Surfactant associated protein D knockout female mice: altered ovarian hormone profile

Surfactant associated protein D (SFTPD, also known as SP-D), a pattern recognition molecule, is an integral component of the mucosal immune system of female reproductive tract (FRT). In addition to host defense functions in the FRT, recent evidences indicate immunomodulatory role of SFTPD in parturition and pre-term labor. Regulation of SFTPD expression by ovarian hormones in the mouse uterus implicates SFTPD of FRT in pregnancy establishment and maintenance. In the current study, we attempted to decipher the functional relevance of SFTPD in FRT by characterizing the fertility parameters of surfactant associated protein D knockout (Sftpd^tm1Jhf/Sftpd^tm1Jhf) female mice. Knockout female mice exhibited extended estrous cycle with altered serum profile of ovarian hormones. We also demonstrate altered expression of ovarian hormone receptors and hormone responsive genes ITGB1, LIF and HOXA10 in uteri of these mice. Knockout females mated with wild type males had significantly smaller litter size due to increased pre-implantation embryo loss. We also observed an altered immune profile in knockout mice uteri with elevated levels of inflammatory cytokines, increased numbers of pro-inflammatory monocytes/macrophages and lower FOXP3 levels during the pre-implantation period. LPS administration to pregnant knockout mice did not result in any increase in embryo implantation loss and was associated with a blunted uterine pro-inflammatory response, plausibly due to higher levels of serum progesterone. Taken together, our results demonstrate that SFTPD deficiency affects female fertility, highlighting roles for SFTPD in ovarian and uterine physiology.     

Developmental roles of tribbles protein family members

The gene tribbles (trbl), identified 12 years ago in genetic screens for mutations that control both cell division and cell migration during embryonic Drosophila development, is the founding member of the Tribbles (Trib) family of kinase‐like proteins that have diverse roles in cell signaling, tissue homeostasis, and cancer. Trib proteins share three motifs: (1) a divergent kinase region (Trib domain) with undetermined catalytic activity, (2) a COP1 site used to direct key target proteins to the proteosome for degradation, and (3) a MEK1 site that binds and modulates MAPKK kinase activity. The notion that Tribs act as scaffolding proteins to balance signaling levels in multiple pathways retains an attractive simplicity, but given recent data showing that divergent kinases act by means of novel catalytic mechanisms, the enzymatic activity of Tribs remains untested. Here, we focus on the role of Tribs during development. Developmental analysis of Drosophila trbl phenotypes reveals tissue‐specific, sometimes contradictory roles. In mammals, multiple Trib isoforms exhibit overlapping and tissue‐specific functions. Recent data indicate the mechanism of Trib activity is conserved and requires the Trib domain. Finally, we discuss the connections between Tribs in disease and cancer that have implications for their normal roles during organogenesis. Developmental Dynamics 241:1239–1248, 2012. © 2012 Wiley Periodicals, Inc.     

Phenylalanine hydroxylase variants interact with the co‐chaperone DNAJC12

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co‐chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild‐type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu^1/1 mouse model, homozygous for the V106A‐Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin‐tagged PAH. This form represented a major fraction of PAH in the Enu^1/1 but was also present in liver of wild‐type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin‐dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.