Animals ; CRISPR-Cas Systems/genetics* ; Fertility/genetics* ; Gene Editing ; Male ; Mice ; Spermatogenesis/genetics* ; Testis

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.
Animals ; CRISPR-Cas Systems/genetics* ; Fertility/genetics* ; Gene Editing ; Humans ; Male ; Mice ; Mice, Knockout ; Testis/metabolism*

Epigenetic factors play an important role in male infertility though about 60%－65% of the disease is idiopathic and its underlying causes are not yet clear. Many studies have indicated that epigenetic modifications, including DNA methylation, histone tail modifications, chromatin remodeling, and non-coding RNAs, may be involved in idiopathic male infertility. Abnormal methylation is associated with decreased sperm quality and fertility. It is known that 1 881 miRNAs are related to male fertility and such non-coding RNAs as piRNA, IncRNA, and circRNA play a regulating role in male reproduction. This review focuses on the value of epigenetics in the etiology and pathogenesis of male infertility, aiming to provide some evidence for the establishment of some strategies for the treatment and prediction of the disease.
DNA Methylation ; Epigenesis, Genetic ; Fertility ; Humans ; Infertility, Male ; genetics ; Male ; MicroRNAs ; physiology ; RNA, Small Interfering ; Spermatozoa

AIMTo evaluate for the first time the frequency of Y chromosome microdeletions and the occurrence of the partial deletions of AZFc region in Moroccan men, and to discuss the clinical significance of AZF deletions.

METHODSWe screened Y chromosome microdeletions and partial deletions of the AZFc region of a consecutive group of infertile men (n = 149) and controls (100 fertile men, 76 normospermic men). AZFa, AZFb, AZFc and partial deletions of the AZFc region were analyzed by polymerase chain reaction (PCR) according to established protocols.

RESULTSAmong the 127 infertile men screened for microdeletion, four subjects were found to have microdeletions: two AZFc deletions and two AZFb+AZFc deletions. All the deletions were found only in azoospermic subjects (4/48, 8.33%). The overall AZFc deletion frequency was low (4/127, 3.15%). AZF microdeletions were not observed in either oligoasthenoteratozoospermia (OATS) or the control. Partial deletions of AZFc (gr/gr) were observed in a total of 7 of the 149 infertile men (4.70%) and 7 partial AZFc deletions (gr/gr) were found in the control group (7/176, 3.98%). In addition, two b2/b3 deletions were identified in two azoospermic subjects (2/149, 1.34%) but not in the control group.

CONCLUSIONOur results suggest that the frequency of Y chromosome AZF microdeletions is elevated in individuals with severe spermatogenic failure and that gr/gr deletions are not associated with spermatogenic failure.

Chromosomes, Human, Y ; diagnostic imaging ; genetics ; Fertility ; Genetic Loci ; Humans ; Infertility, Male ; genetics ; Male ; Morocco ; Reference Values ; Seminal Plasma Proteins ; genetics ; Sequence Deletion ; Spermatogenesis ; genetics ; Ultrasonography

OBJECTIVETo analyze the characteristics of complex chromosomal rearrangement (CCR) in Chinese male carriers and its influence on male fertility.

METHODSUsing the G band technique, we conducted karyotype analysis on the peripheral blood lymphocytes of 1,625 Chinese males with reproductive problems. We also searched CNKI and Wanfang database for CCR-related literature published between January 1984 and November 2013, followed by statistical analysis on the CCR characteristics and reproduction-related data of the CCR carriers.

RESULTSTwo CCR carriers were found among the 1,625 males and another 47 cases identified from the databases. Among the 49 CCR carriers, there were 17 three-way exchange cases (34.7%), 17 double two-way exchange cases (34.7%), and 15 exceptional cases (30.6%), with no statistically significant differences in the incidence of the three types (P > 0.05). Azoospermia- or oligospermia-induced infertility was found in 19 (38.8% ) of the CCR carriers. A total of 87 pregnancies were achieved in the other 30 (61.2%), among which spontaneous abortion occurred in 75.9% (66/87), dead fetus and malformed infant death in 9.2% (8/87), and phenotypically normal offspring in 14.9% (13/87). Recurrent abortion was associated frequently with breakpoints on CCR-involved chromosomes 6, 7, 8, 11, and 16, while dyszoospermia mostly with breakpoints on CCR-involved chromosomes 10 and 14. The breaking occurred more than 3 times at 1p22, 1q25, 2q31, 5p13, 5q35, 6q23, 8q13, and 20p13. Moreo- ver, the breakpoints at 2q31, 5q35, and 8q13 were particularly related to recurrent abortion, while that at 1p22 only to dyszoospermia.

CONCLUSIONCCR is extremely rare. Male CCR carriers are often identified through reproductive problems and have high risks of infertility and abnormal pregnancy and a very low rate of normal newborns. In addition, chromosomes and breakpoints involved in CCR may affect the fertility of male CCR carriers, and some particular chromosomal breakpoints may play a key role in gametogenesis.

Abortion, Habitual ; Azoospermia ; genetics ; Chromosome Aberrations ; Chromosome Banding ; Chromosome Breakpoints ; Female ; Fertility ; genetics ; Heterozygote ; Humans ; Infertility, Male ; genetics ; Karyotyping ; Male ; Oligospermia ; genetics ; Pregnancy ; Reproduction ; Translocation, Genetic

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility. However, the exact mechanisms underlying the behavior of spermatogonia, including spermatogonial stem cell (SSC) self-renewal and spermatogonial proliferation and differentiation, are not fully understood. Recent studies demonstrated that the mTOR complex 1 (mTORC1) signaling pathway plays a crucial role in spermatogonial development, but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined. In this study, we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia. The Mtor knockout (KO) mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age. These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre. Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice, suggesting that spermatogonial differentiation was inhibited. Spermatogonial proliferation was also impaired in Mtor KO mice, leading to a diminished spermatogonial pool and total germ cell population. Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.
Animals ; Cell Proliferation/genetics* ; Fertility/genetics* ; Male ; Mice ; Mice, Knockout ; Spermatogenesis/genetics* ; Spermatogonia/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Testis/metabolism*

Lipid droplets, which are conserved across almost all species, are cytoplasmic organelles used to store neutral lipids. Identification of lipid droplet regulators will be conducive to resolving obesity and other fat-associated diseases. In this paper, we selected 11 candidates that might be associated with lipid metabolism in Caenorhabditis elegans. Using a BODIPY 493/503-based flow cytometry screen, 6 negative and 3 positive regulators of fat content were identified. We selected one negative regulator of lipid content, C13C4.5, for future study. C13C4.5 was mainly expressed in the worm intestine. We found that this gene was important for maintaining the metabolism of lipid droplets. Biochemical results revealed that 50% of triacylglycerol (TAG) was lost in C13C4.5 knockout worms. Stimulated Raman scattering (SRS) signals in C13C4.5 mutants showed only 49.6% of the fat content in the proximal intestinal region and 86.3% in the distal intestinal region compared with wild type animals. The mean values of lipid droplet size and intensity in C13C4.5 knockout animals were found to be significantly decreased compared with those in wild type worms. The LMP-1-labeled membrane structures in worm intestines were also enlarged in C13C4.5 mutant animals. Finally, fertility defects were found in C13C4.5(ok2087) mutants. Taken together, these results indicate that C13C4.5 may regulate the fertility of C. elegans by changing the size and fat content of lipid droplets by interfering with lysosomal morphology and function.
Animals ; Biological Evolution ; Caenorhabditis elegans ; genetics ; growth & development ; metabolism ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Fertility ; Flow Cytometry ; Gene Knockout Techniques ; Humans ; Lipid Metabolism ; genetics ; Lysosomes ; genetics ; metabolism ; Membrane Proteins ; genetics ; Metabolic Networks and Pathways ; genetics ; Triglycerides ; metabolism

OBJECTIVETo investigate the differences in semen quality at different times of reanalysis and the correlation of sperm DNA fragmentation index (DFI) with sperm motility alteration using semen samples completely liquefied and normal in initial examination.

METHODSWe analyzed 127 semen samples up to the inclusion criteria with the computer-assisted semen analysis (CASA) system at 15, 30 and 60 min after semen collection, and obtained sperm morphology parameters and DFI by Shorr staining and acridine orange test (AOT) , respectively.

RESULTSSperm concentration, and the percentages of grades a and b sperm showed no statistically significant differences at the three time points (P > 0.05). The percentages of grades a + b and a + b + c sperm were significantly higher at 15 min than at 30 and 60 min after semen collection (P < 0.05), but with no significant difference between the latter two time points (P > 0.05). The incidence of alternation from normal to abnormal in at least one index of sperm motility at different times was 25.2%, but there were no significant differences in sperm DFI and morphology between the normal and abnormal groups (P > 0.05). Among the altered parameters of sperm motility from 15 to 60 min, the percentages of grades a, a + b and a + b + c sperm were all positively correlated with sperm DFI (P < 0.05).

CONCLUSIONSemen samples completely liquefied within 15 min after collection and normal in initial examination, when reanalyzed at 30 and 60 min, showed significant decreases in the percentages of grades a + b and a + b + c sperm, but not in the percentages of grades a and b sperm, and the parameters of sperm motility might be abnormal. Thus, at least 2 sperm analyses are required for a comprehensive evaluation of fertility. Significant difference between the results of the two analyses, and particularly a markedly reduced percentage of rapidly progressive sperm, might indicate sperm DNA damage, and thus the necessity of sperm DNA damage detection.

Adult ; DNA Damage ; DNA Fragmentation ; Diagnosis, Computer-Assisted ; Fertility ; genetics ; Humans ; Male ; Middle Aged ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa ; Young Adult

The circadian clock has been linked to female reproductive physiology and endocrine in mammals. Epidemiological studies of female shift workers have shown increased rates of abnormal reproduction and adverse pregnancy. But little is known how the circadian rhythms affect reproduction. The aim of the present study was to investigate the influences of circadian rhythms on estrous cycle in female mice using clock gene Rev-erb-α knock out (Rev-erb-α(-/-)) mice. To test the fertility of Rev-erb-α(-/-) mice, litter sizes were counted after mating with C57BL/6J male mice. HE staining was used to observe the change of follicle development. The number of embryos of Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice was compared 1.5 d after mating with C57BL/6J male mice. Then Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice were housed to adult, and daily vaginal lavage with 0.9% saline was used to monitor estrous cycle for at least 30 days. Quantity of various cells was counted on specified smears views after staining. We observed estrous cycles of Rev-erb-α(+/+) and Rev-erb-α(-/-) female mice using line plots and periodic spectrograms. The results showed that the Rev-erb-α(-/-) female mice were infertility, and the number of embryos of Rev-erb-α(-/-) females was less than that of Rev-erb-α(+/+) females. However, the follicle development of Rev-erb-α(-/-) female mice was normal. The estrous cycle of Rev-erb-α(-/-) female mice was 3.22 days longer than that of Rev-erb-α(+/+) female mice. The results suggest that loss of Rev-erb-α prolongs estrous cycle, which is probably one of the reasons for female mice infertility, and circadian rhythm is important for mammalian estrous cycle.
Animals ; Circadian Rhythm ; Estrous Cycle ; Female ; Fertility ; Litter Size ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nuclear Receptor Subfamily 1, Group D, Member 1 ; genetics ; physiology ; Pregnancy

OBJECTIVETo screen and identify differentially expressed genes between a fertile patient and another infertile patient who belonged to a large Chinese pedigree affected with androgen insensitivity syndrome (AIS).

METHODSWe constructed the forward and reversed subtracted libraries using genital skin fibroblasts (GSF), which were obtained from the fertile patient MJ and infertile patient ZGJ, as tester respectively. Candidate clones were screened with colony in situ hybridization, dot blot, and Southern blot analysis step by step and conformed with Northern blot analysis. The potential positive clones were sequenced and the homology of the sequences was analyzed.

RESULTSThe forward and reversed subtracted libraries containing differentially expressed pattern of two GSF cell lines were constructed. Two positive clones identified by Northern blot were obtained in the reversed subtracted library. Eleven candidate clones from the two libraries that failed to hybridize with both RNA populations were obtained simultaneously, which might represent differentially expressed low abundance transcripts. Sequencing results and homology analysis demonstrated that the two positive clones were significantly homologous with the genes of autotaxin-t and calcium binding protein calcyclin (S100A6), respectively.

CONCLUSIONSTwo positive clones and eleven clones showing no hybridization signals may represent differentially expressed genes between the two GSFs. This finding may be useful to elucidate the molecular mechanisms leading to phenotypic variation and preserved fertility of the AIS pedigree.

Androgen-Insensitivity Syndrome ; complications ; genetics ; Blotting, Northern ; Fertility ; genetics ; Fibroblasts ; cytology ; Gene Expression Profiling ; Gene Library ; Genitalia, Male ; cytology ; Humans ; In Vitro Techniques ; Infertility, Male ; etiology ; genetics ; Male ; Nucleic Acid Hybridization ; methods ; Pedigree ; Polymerase Chain Reaction ; Skin ; cytology