SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

BACKGROUND Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which can reconstruct germ cell lineage in mice.However,the application of SSCs for male fertility restoration is hindered due to the unclear mechanisms of proliferation and self-renewal in humans.AIM To investigate the role and mechanism of SPOC domain-containing protein 1(SPOCD1)in human SSC proliferation.METHODS We analyzed publicly available human testis single-cell RNA sequencing(RNAseq)data and found that SPOCD1 is predominantly expressed in SSCs in the early developmental stages.Small interfering RNA was applied to suppress SPOCD1 expression to detect the impacts of SPOCD1 inhibition on SSC proliferation and apoptosis.Subsequently,we explored the target genes of SPOCD1 using RNA-seq and confirmed their role by restoring the expression of the target genes.In addition,we examined SPOCD1 expression in some non-obstructive azoospermia(NOA)patients to explore the correlation between SPOCD1 and NOA.RESULTS The uniform manifold approximation and projection clustering and pseudotime analysis showed that SPOCD1 was highly expressed in the early stages of SSC,and immunohistological results showed that SPOCD1 was mainly localized in glial cell line-derived neurotrophic factor family receptor alpha-1 positive SSCs.SPOCD1 knockdown significantly inhibited cell proliferation and promoted apoptosis.RNA-seq results showed that SPOCD1 knockdown significantly downregulated genes such as adenylate kinase 4(AK4).Overexpression of AK4 in SPOCD1 knockdown cells partially reversed the phenotypic changes,indicating that AK4 is a functional target gene of SPOCD1.In addition,we found a significant downregulation of SPOCD1 expression in some NOA patients,suggesting that the downregulation of SPOCD1 may be relevant for NOA.CONCLUSION Our study broadens the understanding of human SSC fate determination and may offer new theories on the etiology of male infertility.

FOXP4 promotes proliferation of human spermatogonial stem cells

Continuous self-renewal and differentiation of spermatogonial stem cells(SSCs)is vital for maintenance of adult spermatogenesis.Although several spermatogonial stem cell regulators have been extensively investigated in rodents,regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established.We analyzed single-cell sequencing data from the human testis and found that forkhead box P4(FOXP4)expression gradually increased with development of SSCs.Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential.Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis.FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis.These findings imply that FOXP4 is involved in human SSC proliferation,which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors

Aim： To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis （2-DE） reference map of human spermatozoal proteins in a pH range of 3.5-9.0. Methods： In order to reveal more protein spots, immobilized pH gradient strips （24 cm） of broad range of pH 3-10 and the narrower range of pH 6-9, as well as different overlapping narrow range pH immobilized pH gradient （IPG） strips, including 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0 and 5.5-6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. Results： The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3 872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3-10 IPG strip （1 306 spots）. Conclusion： The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility.

A newly discovered mutation in PICK1 in a human with globozoospermia

Globozoospermia is a human infertility syndrome caused by spermatogenesis defects （OMIM 102530）. Acrosome plays an important role at the site of sperm-zonapellucida binding during the fertilization process. Thus, malformation of the acrosome is the most prominent feature seen in globozoospermia. Disruption of several mouse genes, including Gopc （Golgi-associated PDZ and coiled-coil motif containing protein）, Hrb （HIV-I Rev binding protein）, Csnk2α2 （casein kinase 2, α prime polypeptide） and Pick1 （protein interacting with C kinase 1）, results in a phenotype similar to globozoospermia in humans, which suggests their potential role in the disease. However, no mutations with a clear link to globozoospermia have been identified in these genes in humans. In this study, we screened the candidate genes men- tioned above in three globozoospermia type I patients and discovered a homozygous missense mutation （G198A） in exon 13 of the PICK1 gene in a Chinese family. The family member affected by this homozygous missense mutation showed a complete lack of acrosome. Using the candidate gene screening strategy, our study is the first to identify an autosomal recessive genetic mutation in PICK1 that was responsible for globozoospermia in humans.

Phenotypic and molecular characteristics of androgen insensitivity syndrome patients

Androgen insensitivity syndrome （AIS）, an X-linked recessive genetic disorder of sex development, is caused by mutations in the androgen receptor （AR） gene, and is characterized by partial or complete inability of specific tissues to respond to androgens in individuals with the 46,XY karyotype. This study aimed to investigate AR gene mutations and to characterize genotype-phenotype correlations. Ten patients from unrelated families, aged 2-31 years, were recruited in the study. Based on karyotype, altered hormone profile, and clinical manifestations, nine patients were preliminarily diagnosed with complete AIS and one with partial AIS. Genetic analysis of AR gene revealed the existence of 10 different mutations, of which five were novel （c.2112 C〉G[p.STO4R], c.2290T〉A[p.Y764N], c.2626C〉T[p.Q876X], c.933dupC[p.K313Qfs＊28], and c.1067delC[p.A356Efs＊123]）; the other five were previously reported （c.1789G〉A[p.A597T], c.2566C〉T[p.R856C], c.2668G〉A[p.V890M], c.2679C〉T[p.P893L], and c.1605C〉G[p.Y535X]）. Regarding the distribution of these mutations, 60.0% were clustered in the ligand-binding domain of AR gene. Exons 1 and 8 of AR gene each accounted for 30.0% （3/10） of all mutations. Most of the truncation mutations were in exon 1 and missense mutations were mainly located in exons 4-8. Our study expands the spectrum of AR gene mutations and confirms the usefulness of AR gene sequencing to support a diagnosis of AIS and to enable prenatal or antenatal screening.

Prenatal diagnosis of nonmosaic tetrasomy 9p by microdissection and FISH:case report

Marker chromosome （mar） is a structurally abnormal chromosome that can not be identified with conventional cytogenetic techniques （ISCN, 2005）. The incidence of supernumerary marker chromosomes （SMCs） found at prenatal diagnosis varies from 0.4/1000 to 1.5/1000. Their genetic effects may range from harmless to detrimental, and in de novo cases, the rate of phenotypic abnormalities is statistically increased. Therefore, identification of SMCs has become imperative in prenatal diagnosis.

PLZF^posc-KIT^pos-delineated A1-A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation

While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2–A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.

Non-invasive prenatal molecular detection of a fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR

Conventional prenatal diagnosis relies on invasive chorionic biopsy or amniocentesis, which increases the risk of miscarriage, and is undertaken at 11-20 weeks gestation.1 The discovery of cell-free fetal DNA in maternal plasma has, however, offered a new strategy for non-invasive prenatal diagnosis.2 Cell-free fetal DNA in maternal plasma has been used for the determination of fetal gender3 and RHD status4 as well as testing certain monogenic diseases such as 13-thalassemia5 and cystic fibrosis.6 However,

A rare polypyrimidine tract mutation in the androgen receptor gene results in complete androgen insensitivity syndrome

Dear Editor, Androgen insensitivity syndrome （AIS） is a common 46, XY disorder of sex development resulting from androgen resistance. AIS can be subdivided into three phenotypes according to the degree of external genital defects： complete AIS （CAIS）, with typical female external genitalia; partial AIS, with predominantly male or ambiguous external genitalia; and mild AIS, with typical male external genitalia. CAIS is the classical manifestation of AIS. Individuals affected by CAIS typically exhibit inguinal swellings during infancy or primary amenorrhea during puberty） AIS is usually caused by mutations in the androgen receptor （AR） gene.

Study of the Sperm Chromosomal Aneuploidies of Isolated Teratozoospermic Men

Objective To determine whether patients with isolated teratozoospermia have increased or decreased incidence of chromosomal aneuploidies. Methods Sperm obtained from isolated teratozoospermic men （teratozoospermic group, n=18） and normal fertile men （the control, n=5） were analyzed using FISH （for chromosomes 18, X and Y）. Results A total of 58 178 spermatozoa were counted from the teratozoospermia group and 16 369 spermatozoa were counted from the control, with the hybridization rates of 97.5% and 98.3%, respectively. The major types of chromosomal aneuploidies were disomy （YY18, XX18, XY18, Y1818 and 3（1818） and diploidy （1818XX, 1818YY, 1818XY）. In the teratozoospermic group and the control, the disomy rates of 18 chromosome were 0.29 ±0.16% and 0.03 ±0.02%, the disomy rates of sex chromo- some were 0.65 ±0.24% and 0.05 ± 0.02%, the diploidy rates were 0.14 ± 0.12% and 0. 04±0.03%, respectively..411 the differences between these two groups were significant （P〈0. 05）.Conclusion Sperm of isolated teratozoospermic men have higher rates of 18, X and Y chromosomal aneuploidies than that of the fertile controls.

Novel biallelic PCNT deletion causing microcephalic osteodysplastic primordial dwarfism type II with congenital heart defect

Dear Editor, Microcephalic osteodysplastic primordial dwarfism type Ⅱ (MOPD Ⅱ )is characterized by developmental retardation, wherein the affected individuals usually present with intrauterine growth retardation and preterm birth (Majewski et al.,1982;Willems et al.,2010).This leads to an average weight of <1,500g at birth and extremely restricted postnatal growth (Hall et al.,2004;Rauch,2011).Clinical manifestations ofMOPD Ⅱ include microcephaly,disproportionately short stature,mild skeletal dysplasia,unusual facial features including a prominent nose,prominent eyes in infancy and early childhood,some affected individuals exhibit slightly reduced intellectual development and cerebral vascular malformations (Willems et al.,2010;Li et al.,2015;Sam et al.,2015).

Factors Relating with Fertilization Failure in Conventional IVF-ET Cycles

Objective To analyze the factors relating with fertilization failure in conventional IVF cycles. Methods The total fertilization failure rate of 2 429 IVF cycles from January 2011 to May 2012 in the Reproductive Centre of the Women and Children Hospital of Jiangxi Province were retrospectively analyzed. Risk factors were identified by univariate and multivariate logistic regression analyses. Results The total fertilization failure rate of conventional IVF-ET rate was 5. 7% （139/ 2 429）. Thepercentage of morphologically normal sperm （11.1 ±5.8% vs 13.4 ±5.3%）, progressive motility （47.4± 10.5% vs 50.1 ±8.6%）, percentage of couple primary infertility （79.1% vs 44.3%）, percentage o f female primary infertility （69.1% vs 36.3%）, percentage of male primary infertility （74.8% vs 41.7%）, percentage of without oviduct obstruction patients （30.2% vs 13.6%） and percentage of couples primary infertility （79. 1% vs 44. 3%） of the fertilization failure patients were significantly different from those of the fertilized patients （P〈O.05）. Besides, higher total fertilization failure rate was found in couples with isolated teratozoospermia than in couples with normal percentage of morphologically normal sperm （15.0% vs 5.2%）. After that, it was found by multivariate logistic regression analyses that many factors including percentage of morphologically normal sperm, sperm concentration, couples primary infertility and female infertility years were related with fertilization failure. Conclusion Patients with low percentage of morphologically normal sperm （〈4%）, low sperm concentration and motility, male primary infertility, female primary infertility, without oviduct obstruction, long female infertility years or/and couples primary infertility are at high risk of fertilization failure. More attention should be paid to these patients for avoiding the fertilization failure.