Splicing mutation in Sbf1 causes nonsyndromic male infertility in the rat

In the inbred SHR/OlaIpcv rat colony, we identified males with small testicles and inability to reproduce. By selectively breeding their parents, we revealed the infertility to segregate as an autosomal recessive Mendelian character. No other phenotype was observed in males, and females were completely normal. By linkage using a backcross with Brown Norway strain, we mapped the locus to a 1.2Mbp segment on chromosome 7, harboring 35 genes. Sequencing of candidate genes revealed a G to A substitution in a canonical ‘AG’ splice site of intron 37 in Sbf1 (SET binding factor 1, alias myotubularin-related protein 5). This leads to either skipping exon 38 or shifting splicing one base downstream, invariantly resulting in frameshift, premature stop codon and truncation of the protein. Western blotting using two anti-Sbf1 antibodies revealed absence of the full-length protein in the mutant testis. Testicles of the mutant males were significantly smaller compared with SHR from 4weeks, peaked at 84% wild-type weight at 6weeks and declined afterward to 28%, reflecting massive germ cell loss. Histological examination revealed lower germ cell number; latest observed germ cell stage were round spermatids, resulting in the absence of sperm in the epididymis (azoospermia). SBF1 is a member of a phosphatase family lacking the catalytical activity. It probably modulates the activity of a phosphoinositol phosphatase MTMR2. Human homozygotes or compound heterozygotes for missense SBF1 mutations exhibit Charcot–Marie–Tooth disease (manifested mainly as progressive neuropathy), while a single mouse knockout reported in the literature identified male infertility as the only phenotype manifestation.

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Protective role of α-actinin-3 in the response to an acute eccentric exercise bout

Journal of Applied Physiology ◽

10.1152/japplphysiol.01007.2009 ◽

2010 ◽

Vol 109 (2) ◽

pp. 564-573 ◽

Cited By ~ 46

Author(s):

Barbara Vincent ◽

An Windelinckx ◽

Henri Nielens ◽

Monique Ramaekers ◽

Marc Van Leemputte ◽

...

Keyword(s):

Muscle Damage ◽

Eccentric Exercise ◽

Stop Codon ◽

Premature Stop Codon ◽

Exercise Bout ◽

Cell Number ◽

Protective Role ◽

Structural Function ◽

Marker Genes

The ACTN3 gene encodes for the α-actinin-3 protein, which has an important structural function in the Z line of the sarcomere in fast muscle fibers. A premature stop codon (R577X) polymorphism in the ACTN3 gene causes a complete loss of the protein in XX homozygotes. This study investigates a possible role for the α-actinin-3 protein in protecting the fast fiber from eccentric damage and studies repair mechanisms after a single eccentric exercise bout. Nineteen healthy young men (10 XX, 9 RR) performed 4 series of 20 maximal eccentric knee extensions with both legs. Blood (creatine kinase; CK) and muscle biopsy samples were taken to study differential expression of several anabolic (MyoD1, myogenin, MRF4, Myf5, IGF-1), catabolic (myostatin, MAFbx, and MURF-1), and contraction-induced muscle damage marker genes [cysteine- and glycine-rich protein 3 (CSRP3), CARP, HSP70, and IL-6] as well as a calcineurin signaling pathway marker (RCAN1). Baseline mRNA content of CSRP3 and MyoD1 was 49 ± 12 and 67 ± 25% higher in the XX compared with the RR group ( P = 0.01–0.045). However, satellite cell number was not different between XX and RR individuals. After eccentric exercise, XX individuals tended to have higher serum CK activity ( P = 0.10) and had higher pain scores than RR individuals. However, CSRP3 ( P = 0.058) and MyoD1 ( P = 0.08) mRNA expression tended to be higher after training in RR individuals compared with XX α-actinin-3-deficient subjects. This study suggests a protective role of α-actinin-3 protein in muscle damage after eccentric training and an improved stress-sensor signaling, although effects are small.

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Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

Endocrinology ◽

10.1210/en.2011-1106 ◽

2011 ◽

Vol 152 (10) ◽

pp. 3975-3985 ◽

Cited By ~ 12

Author(s):

Rebecca M. Harris ◽

Jeffrey Weiss ◽

J. Larry Jameson

Keyword(s):

Male Infertility ◽

Germ Cell ◽

Missense Mutation ◽

Cell Cycle Progression ◽

Cell Loss ◽

Kinase Domain ◽

Heterozygous Mutation ◽

Male Hypogonadism ◽

Testis Size ◽

Pachytene Spermatocytes

The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis.

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Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer

BMC Medicine ◽

10.1186/s12916-020-01844-y ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Melissa D. Tharmalingam ◽

Gabriele Matilionyte ◽

William H. B. Wallace ◽

Jan-Bernd Stukenborg ◽

Kirsi Jahnukainen ◽

...

Keyword(s):

Germ Cell ◽

Childhood Cancer ◽

Cell Loss ◽

Testicular Tissue ◽

Cell Number ◽

Human Testis ◽

Cell Populations ◽

Similar Degree ◽

Future Fertility ◽

The Impact

Abstract Background Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues. Methods We developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24–240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified. Results Cisplatin exposure resulted in a significant reduction in the total number of germ cells (− 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (− 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (− 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure. Conclusions This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

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Gene Expression in Brown Norway Rat Leydig Cells: Effects of Age and of Age-Related Germ Cell Loss

Endocrinology ◽

10.1210/endo.142.12.8526 ◽

2001 ◽

Vol 142 (12) ◽

pp. 5277-5285 ◽

Cited By ~ 15

Author(s):

Patrick Syntin ◽

Haolin Chen ◽

Barry R. Zirkin ◽

Bernard Robaire

Keyword(s):

Gene Expression ◽

Germ Cell ◽

Leydig Cells ◽

Cell Loss ◽

Brown Norway ◽

Norway Rat ◽

Age Related ◽

Brown Norway Rat

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TRIM71 deficiency causes germ cell loss during mouse embryogenesis and promotes human male infertility

10.1101/2021.02.01.429172 ◽

2021 ◽

Author(s):

Lucia A. Torres-Fernández ◽

Jana Emich ◽

Yasmine Port ◽

Sibylle Mitschka ◽

Marius Wöste ◽

...

Keyword(s):

Male Infertility ◽

Germ Cell ◽

Germ Cells ◽

Cell Loss ◽

Seminiferous Tubules ◽

Obstructive Azoospermia ◽

Sequencing Analysis ◽

Mouse Embryogenesis ◽

Human Male

AbstractMutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling the complex molecular network orchestrating germ cell development. TRIM71 is a stem cell-specific factor essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype, which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of developing germ cells in the testes’ seminiferous tubules. Infertility originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, in vitro growth competition assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells, a human GCT-derived cell line which we used as a surrogate model for proliferating PGCs, showed that TRIM71 promotes NCCIT cell proliferation and survival. Our data collectively suggest that germ cell loss in cKO mice results from combined defects during the specification and maintenance of PGCs prior to their sex determination in the genital ridges. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with SCO phenotype. Our work reveals for the first time an association of TRIM71 variants with human male infertility, and uncovers further developmental roles for TRIM71 in the generation and maintenance of germ cells during mouse embryogenesis.

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The Role of DND1 in Cancers

Cancers ◽

10.3390/cancers13153679 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3679

Author(s):

Yun Zhang ◽

Jyotsna D. Godavarthi ◽

Abie Williams-Villalobo ◽

Shahrazad Polk ◽

Angabin Matin

Keyword(s):

Germ Cell ◽

Translational Regulation ◽

Rna Binding ◽

Human Cancer ◽

Stop Codon ◽

Primordial Germ Cell ◽

Germ Cell Tumors ◽

Premature Stop Codon ◽

Protein Activity ◽

Testicular Germ Cell

The Ter mutation in Dead-End 1 (Dnd1), Dnd1Ter, which leads to a premature stop codon, has been determined to be the cause for primordial germ cell deficiency, accompanied with a high incidence of congenital testicular germ cell tumors (TGCTs) or teratomas in the 129/Sv-Ter mice. As an RNA-binding protein, DND1 can bind the 3′-untranslated region (3′-UTR) of mRNAs and function in translational regulation. DND1 can block microRNA (miRNA) access to the 3′-UTR of target mRNAs, thus inhibiting miRNA-mediated mRNA degradation and up-regulating translation or can also function to degrade or repress mRNAs. Other mechanisms of DND1 activity include promoting translation initiation and modifying target protein activity. Although Dnd1Ter mutation causes spontaneous TGCT only in male 129 mice, it can also cause ovarian teratomas in mice when combined with other genetic defects or cause germ cell teratomas in both genders in the WKY/Ztm rat strain. Furthermore, studies on human cell lines, patient cancer tissues, and the use of human cancer genome analysis indicate that DND1 may possess either tumor-suppressive or -promoting functions in a variety of somatic cancers. Here we review the involvement of DND1 in cancers, including what appears to be its emerging role in somatic cancers.

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Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650386 ◽

1996 ◽

Vol 75 (06) ◽

pp. 870-876 ◽

Cited By ~ 4

Author(s):

José Manuel Soria ◽

Lutz-Peter Berg ◽

Jordi Fontcuberta ◽

Vijay V Kakkar ◽

Xavier Estivill ◽

...

Keyword(s):

Splice Site ◽

Protein C ◽

Stop Codon ◽

Premature Stop Codon ◽

Allelic Exclusion ◽

Polymorphism Analysis ◽

Type I ◽

Protein C Deficiency ◽

Nonsense Mutations ◽

Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

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