Sperm Nuclear Maturity Research Articles

Biallelic variants in KCTD19 associated with male factor infertility and oligoasthenoteratozoospermia.

Can whole-exome sequencing (WES) reveal new genetic factors responsible for male infertility characterized by oligozoospermia? We identified biallelic missense variants in the Potassium Channel Tetramerization Domain Containing 19 gene (KCTD19) and confirmed it to be a novel pathogenic gene for male infertility. KCTD19 is a key transcriptional regulator that plays an indispensable role in male fertility by regulating meiotic progression. Kctd19 gene-disrupted male mice exhibit infertility due to meiotic arrest. We recruited a cohort of 536 individuals with idiopathic oligozoospermia from 2014 to 2022 and focused on five infertile males from three unrelated families. Semen analysis data and ICSI outcomes were collected. WES and homozygosity mapping were performed to identify potential pathogenic variants. The pathogenicity of the identified variants was investigated in silico and in vitro. Male patients diagnosed with primary infertility were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Genomic DNA extracted from affected individuals was used for WES and Sanger sequencing. Sperm phenotype, sperm nuclear maturity, chromosome aneuploidy, and sperm ultrastructure were assessed using hematoxylin and eosin staining and toluidine blue staining, FISH and transmission electron microscopy. The functional effects of the identified variants in HEK293T cells were investigated via western blotting and immunofluorescence. We identified three homozygous missense variants (NM_001100915, c.G628A:p.E210K, c.C893T:p.P298L, and c.G2309A:p.G770D) in KCTD19 in five infertile males from three unrelated families. Abnormal morphology of the sperm heads with immature nuclei and/or nuclear aneuploidy were frequently observed in individuals with biallelic KCTD19 variants, and ICSI was unable to rescue these deficiencies. These variants reduced the abundance of KCTD19 due to increased ubiquitination and impaired its nuclear colocalization with its functional partner, zinc finger protein 541 (ZFP541), in HEK293T cells. The exact pathogenic mechanism remains unclear, and warrants further studies using knock-in mice that mimic the missense mutations found in individuals with biallelic KCTD19 variants. Our study is the first to report a likely causal relationship between KCTD19 deficiency and male infertility, confirming the critical role of KCTD19 in human reproduction. Additionally, this study provided evidence for the poor ICSI clinical outcomes in individuals with biallelic KCTD19 variants, which may guide clinical treatment strategies. This work was supported by the National Key Research and Developmental Program of China (2022YFC2702604 to Y.-Q.T.), the National Natural Science Foundation of China (81971447 and 82171608 to Y.-Q.T., 82101961 to C.T.), a key grant from the Prevention and Treatment of Birth Defects from Hunan Province (2019SK1012 to Y.-Q.T.), a Hunan Provincial Grant for Innovative Province Construction (2019SK4012), and the China Postdoctoral Science Foundation (2022M721124 to W.W.). The authors declare no conflicts of interest. N/A.

Protective Effects of Sesamin on Cytoxan-Induced Spermatogenesis Dysfunction by Regulating RNF8-ubH2A/ubH2B Pathways in Male Mice.

Most of the clinically infertile patients show spermatogenesis dysfunction. Cyclophosphamide, as an anticancer drug, can induce spermatogenesis dysfunction. Sesamin is the main bioactive component of natural lignans in sesame. It is abundant in sesame oil and has strong biological activities such as antioxidant, antibacterial, and hypoglycemic properties. By establishing the model of spermatogenic dysfunction induced by cyclophosphamide in male mice and then feeding sesamin (50, 100, and 200 mg/kg) for 2 weeks, we proved that sesamin can improve the reproductive organ damage induced by cyclophosphamide and increase the number and activity of sperms. Sesamin can resist cyclophosphamide-induced sperm nuclear maturity and DNA damage by increasing the expression levels of histones H2A and H2B in the testis. In addition, sesamin can improve the ubiquitination of histones regulated by RNF8 to protect the testis. In conclusion, these results suggest that sesamin can improve spermatogenic dysfunction induced by cyclophosphamide, which may be mediated by ubiquitination of histones.

Sperm nuclear maturity and chromatin stability in subfertile patients: Density gradient centrifugation is fair but non-discriminative in selecting the right population

Multiple technologies exploring chromatin structure anomalies have been applied during the last decade to evaluate fertility disorders and to increase the predictive value of sperm analysis for procreation in vivo and in vitro. Our aim was to implement sperm nuclear maturity and nuclear chromatin stability as a functional test for male infertility diagnosis and to compare it with a fertile group. As semen processing is an integral part of assisted reproductive technologies the impact of density gradient centrifugation in selecting sperm based on nuclear maturity and stability was also analyzed. Flow cytometry combined with fluorescent dyes exhibiting affinity for DNA was implemented. Both nuclear parameters correlated significantly with semen parameters. The control fertile group had significantly higher mean condensed population and a significantly lower hypocondensed and hypercondensed fractions as compared to the subfertile study group. Density gradient centrifugation succeeded in selecting the condensed population in both the control and study groups, while reducing the hypocondensed percentage. The hypercondensed population which was ten-fold higher in the study group remained unchanged after selection, in both the control and the study groups. Sperm nuclear maturity and chromatin stability appears to be homogenous in the fertile sperm donors and heterogeneous in subfertile patients. Sperm preparation for assisted reproduction should aim to minimize the risk of abnormal spermatozoa being used for fertilization.

WITHDRAWN: Protocatechualdehyde improves antineoplastic cyclophosphamide-induced mouse testicular damage

// Chun-Na An 1 , Xiao-Ping Pu 2 , Qiu-Ping Yang 3 , Jing-Ting Dong 3 , Shu-Ying Han 1 , Rui Wei 1 and Hong-Ning Zhang 4 1 Department of Pharmacology, School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, China 2 Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China 3 Jitang College, North China University of Science and Technology, Tangshan, China 4 Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing, China Correspondence to: Hong-Ning Zhang, email: zhang_hn2008@126.com Keywords: protocatechualdehyde; cyclophosphamide; testicular damage; DJ-1 Received: July 21, 2017      Accepted: November 28, 2017      Epub: January 02, 2018 ABSTRACT We studied the effects of protocatechualdehyde on mouse sperm survival and sperm membrane integrity in vitro and on cyclophosphamide-induced mouse testicular damage in vivo . Protocatechualdehyde (0.01, 0.1, and 1 mg/mL) improved sperm survival rate and sperm membrane integrity in separated mouse sperm (all P < .05). In cyclophosphamide-treated male mice (60 mg/kg/d by intraperitoneal injection, 5 days of treatment), protocatechualdehyde (40 mg/kg/d by intragastric gavage, 35 days of treatment) increased the testis index, epididymis index, and sperm nuclear maturity (all P < .05). Protocatechualdehyde also improved testis morphology characterized by orderly arranged layers of spermatogenic cells, numbers of sperm in the lumen, normal mesenchymal cells, and close and tidy arrangement of the seminiferous tubules. Protocatechualdehyde also increased testicular superoxide dismutase activity and elevated DJ-1 expression. In addition, it decreased expression of ICAM-1 and enhanced the expression of VCAM-1, PEDF, VEGF, and PPARγ. These findings indicate that protocatechualdehyde increases mouse sperm survival and sperm membrane integrity in vitro and reduces cyclophosphamide-induced mouse testicular damage via DJ-1 and other targets in vivo . Protocatechualdehyde may thus be useful for treating reproductive damage and antineoplastic cyclophosphamide-induced reproductive toxicity.

Detection of single nucleotide polymorphism of holandric genes and association with sperm nuclear maturity in bovines

Implementation of a successful cattle breeding program relies on selection of genetically superior and tested males against various andrological anomalies. In the present project, single stranded conformation polymorphism (SSCP) of holandric genes viz. ubiquitin specific protease, Y-linked (USPY) and RNA binding motif Y-linked (RBMY) was studied for associating with spermatological attributes (sperm chromatin maturity, semen volume, initial motility) in cattle (10 HF crossbred) and buffalo (10 Murrah). The SSCP band patterns revealed species specific variation only in two primer-pairs (US2 and US3) of USPY gene. The maximum and minimum values of percent immature sperm were ranging between 0.392 to 2.586 in cattle and from 0.901 to 4.000 in buffalo. The per cent immature spermatozoa in the ejaculates did not differ significantly either for species or for the categories (acceptable and unacceptable semen donors). The average immature sperm count was the least in buffalo ejaculates (1.383 ± 0.176). The correlation coefficient was found negative between the spermatological parameters (volume and initial motility) and the immature sperm yield in both cattle and buffalo ejaculates. It indicates that the immature sperm production might be negatively associated with semen volume and initial motility of the spermatozoa.

Initial exploration of epididymal sperm nuclear maturity in obstructive azoospermia patients

To ascertain the epididymal sperm nuclear maturity in obstructive azoospermia (OA) patients. A total of 81 infertile males from andriatry clinic of Renji hospital were selected, including 32 OA patients (OA group) and 49 asthenospermia patients (asthenospermia group). Another 32 fertile males were recruited for the control group. All semen samples of the asthenospermia and control groups underwent computer aided semen analysis (CASA). For the OA group, sperm specimens were collected by percutaneous epididymal sperm aspiration (PESA) and semen analyses completed by manual method. Further acridine orange test and aniline blue stain assay were performed. The results were processed with SPSS 15.0. No statistically significant differences existed in semen volume and sperm density between the asthenospermia and control groups (both P > 0.05). The OA group was significantly lower than the control group in sperm density (P < 0.01). The asthenospermia and OA groups were also significantly lower than the control group in sperm motility (both P < 0.01). As to the percentage of grade a + b sperm, the asthenospermia group was also significantly lower than the control group (P < 0.01). Compared with the control group (81% ± 9%, 85% ± 8%), the negative rate of acridine orange test and aniline blue assay of OA (57% ± 20%, 64% ± 20%) and asthenospermia group (62% ± 19%, 67% ± 15%) were statistically lower (all P < 0.05). The sperm nuclear maturity is lower in the asthenospermia and OA groups than that in the control group. Male fertility should be further improved.

Cadmium‐induced infertility in male mice

The effects of cadmium in a concentration similar to that found in Maharloo Lake (Shiraz, Iran) on male reproductive system was studied in adult Balb/c male mice that received 0, 23, and 50 mg/kg of cadmium chloride in 0.5 mL distilled water for 45 days. Sperm count and motility, sperm nuclear maturity and chromatin structure tests were carried out. Testis of each mouse was examined histologically. The treated male mice were mated with females. Prostatic and nonprostatic acid phosphatase activity in blood serum, testis, and prostate, lipid peroxidation and cadmium accumulation in testis, seminal vesicle, and middle 1/3 of the quadericeps femoris muscle were measured. The sperm count, sperm motility, sperm maturity, and the level of testosterone decreased significantly in the high dose adminstered group. Histological studies showed a severe necrosis and atrophy in the testis of high dose group, consequently, there was no successful mating in some groups. The number of newborns and their weights and crown rump lengths reduced. Cadmium accumulation in testis and middle of the quadriceps femoris muscle was significantly higher in animals receiving 50 mg/kg cadmium chloride. Nonprostatic acid phosphatase activity decreased, whereas prostatic acid phosphatase activity increased significantly in serum of animals receiving 50 mg/kg cadmium chloride. Also prostatic acid phosphatase activity decreased significantly in prostate of animals receiving 50 mg/kg cadmium chloride. Lipid peroxidation was significantly higher in testis of animals treated with 50 mg/kg cadmium chloride compared with control group. Cadmium affects male reproductive system activity and can cause infertility in mice as an animal model.

Relation between different human sperm nuclear maturity tests and in vitro fertilization.

To consider the relationship between different sperm nuclear maturity tests and in vitro fertilization (IVF) rate, in order to select the most sensitive, specific, and independent factor(s) for prediction of in vitro fertilization. Infertile couples (101) were randomly selected from IVF candidates referred to Isfahan Fertility and Infertility center. Semen samples were collected on the day of oocyte recovery. Following routine semen analysis, major portion of the semen was prepared for routine IVF insemination and the remaining was used for following sperm nuclear maturity tests: chromomycin A3 (CMA3), aniline blue, sodium dodecyl sulphate (SDS) test, and acridine orange test with or without heat shock (87 degrees C, 5 min). Sperms (200) were evaluated for each test. The results were recorded and analyzed for their correlation to fertilization rate, using correlation coefficient, logistic regression analysis, student t-test, and receiver operating characteristics (ROC) curve. Among these tests, aniline blue and CMA3, and semen parameters, sperm morphology, and sperm motility showed a significant correlation with fertilization rate. Using logistic regression analysis, sperm morphology and CMA3 were the only independent factors related to in vitro fertilization. ROC curves showed that among above tests, CMA3 is the most specific and sensitive for sperm nuclear maturity. Among CMA3, aniline blue, SDS test, and acridine orange, CMA3 was the most sensitive and specific test that can be used along with routine semen analysis for more precise prediction of fertilization rate.

Sperm nuclear maturity in spinal cord-injured men: Evaluation by acidic aniline blue stain

For many of the spinal cord-injured (SCI) men who are able to produce an ejaculate, infertility because of poor semen quality is a concern. Impaired spermatogenesis has been attributed as a possible reason for the poor semen quality. If so, events that occur during spermatogenesis may be used as a marker to evaluate the extent of spermatogenic alteration. During spermatogenesis, when the sperm nuclear condensation occurs, lysine-rich somatic histone are replaced by arginine-rich protamines in the DNA. Acidic aniline blue preferentially stains the immature sperm nucleus blue by binding to the lysine. Hence, each sperm can be individually evaluated for nuclear maturity. To test this concept, the nuclear maturity of sperm from 12 SCI men obtained by vibratory stimulation was compared with sperm samples obtained by self-masturbation from 104 non-SCI men. Sperm smears stained with acidic aniline blue were evaluated for nuclear maturity. The percent of unstained spermatozoa for non-SCI men (mean ± SEM; 83.4 ± 1.1%) was not statistically different from that of the SCI men (79.7 ± 4.8%). However, the sperm motility (70.5 ± 1.2%) and the percentage of normal sperm morphology (50.8 ± 0.7%) of non-SCI men were significantly ( P < .01) different from those of the SCI men (36.5 ± 6.8% and 44.0 ± 2.4%). It seems that the poor semen quality observed in SCI men is probably not caused by inadequate nuclear maturity of the spermatozoa.

Evaluation of sperm nuclear maturity with acidic aniline blue in spinal cord injured men

Evaluation of sperm nuclear maturity with acidic aniline blue in spinal cord injured men

Evaluation of sperm nuclear maturity with acidic aniline blue in spinal cord injured men

Evaluation of sperm nuclear maturity with acidic aniline blue in spinal cord injured men