Multiple morphological abnormalities of the flagella (MMAF) represent a severe form of sperm defects leading to asthenozoospermia and male infertility. In this study, we identified a novel homozygous splicing mutation (c.871-4 ACA>A) in the adenylate kinase 7 (AK7) gene by whole-exome sequencing in infertile individuals. Spermatozoa from affected individuals exhibited typical MMAF characteristics, including coiled, bent, short, absent, and irregular flagella. Transmission electron microscopy analysis showed disorganized axonemal structure and abnormal mitochondrial sheets in sperm flagella. Immunofluorescence staining confirmed the absence of AK7 protein from the patients' spermatozoa, validating the pathogenic nature of the mutation. This study provides direct evidence linking the AK7 gene to MMAF-associated asthenozoospermia in humans, expanding the mutational spectrum of AK7 and enhancing our understanding of the genetic basis of male infertility.
Humans ; Male ; Sperm Tail/ultrastructure* ; Homozygote ; Consanguinity ; Asthenozoospermia/pathology* ; Infertility, Male/genetics* ; Mutation ; Pakistan ; Adenylate Kinase/genetics* ; Adult ; Pedigree ; RNA Splicing ; Exome Sequencing ; Spermatozoa

Male infertility can result from impaired sperm motility caused by multiple morphological abnormalities of the flagella (MMAF). Distinct projections encircling the central microtubules of the spermatozoal axoneme play pivotal roles in flagellar bending and spermatozoal movement. Mammalian sperm-associated antigen 17 ( SPAG17 ) encodes a conserved axonemal protein of cilia and flagella, forming part of the C1a projection of the central apparatus, with functions related to ciliary/flagellar motility, skeletal growth, and male fertility. This study investigated two novel homozygous SPAG17 mutations (M1: NM_206996.2, c.829+1G>T, p.Asp212_Glu276del; and M2: c.2120del, p.Leu707*) identified in four infertile patients from two consanguineous Pakistani families. These patients displayed the MMAF phenotype confirmed by Papanicolaou staining and scanning electron microscopy assays of spermatozoa. Quantitative real-time polymerase chain reaction (PCR) of patients' spermatozoa also revealed a significant decrease in SPAG17 mRNA expression, and immunofluorescence staining showed the absence of SPAG17 protein signals along the flagella. However, no apparent ciliary-related symptoms or skeletal malformations were observed in the chest X-rays of any of the patients. Transmission electron microscopy of axoneme cross-sections from the patients showed incomplete C1a projection and a higher frequency of missing microtubule doublets 1 and 9 compared with those from fertile controls. Immunofluorescence staining and Western blot analyses of spermatogenesis-associated protein 17 (SPATA17), a component of the C1a projection, and sperm-associated antigen 6 (SPAG6), a marker of the spring layer, revealed disrupted expression of both proteins in the patients' spermatozoa. Altogether, these findings demonstrated that SPAG17 maintains the integrity of spermatozoal flagellar axoneme, expanding the phenotypic spectrum of SPAG17 mutations in humans.
Humans ; Male ; Infertility, Male/pathology* ; Sperm Tail/ultrastructure* ; Homozygote ; Microtubule-Associated Proteins/genetics* ; Axoneme/genetics* ; Spermatozoa/ultrastructure* ; Adult ; Mutation ; Sperm Motility/genetics* ; Pedigree ; Microtubules ; Microtubule Proteins/genetics*

The syndrome of multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most serious kinds of sperm defects, leading to asthenoteratozoospermia and male infertility. In this study, we use whole-exome sequencing to identify genetic factors that account for male infertility in a patient born from a consanguineous Pakistani couple. A homozygous frameshift mutation (c.1399_1402del; p.Gln468ArgfsTer2) in axonemal dynein light chain domain containing 1 ( AXDND1 ) was identified in the patient. Sanger sequencing data showed that the mutation was cosegregated recessively with male infertility in this family. Papanicolaou staining and scanning electron microscopy analysis of the sperm revealed severely abnormal flagellar morphology in the patient. Immunofluorescence and western blot showed undetectable AXDND1 expression in the sperm of the patient. Transmission electron microscopy analysis showed disorganized sperm axonemal structure in the patient, particularly missing the central pair of microtubules. Immunofluorescence staining showed the absence of sperm-associated antigen 6 (SPAG6) and dynein axonemal light intermediate chain 1 (DNALI1) signals in the sperm flagella of the patient. These findings indicate that AXDND1 is essential for the organization of flagellar axoneme and provide direct evidence that AXDND1 is a MMAF gene in humans, thus expanding the phenotypic spectrum of AXDND1 frameshift mutations.
Humans ; Male ; Sperm Tail/ultrastructure* ; Frameshift Mutation ; Infertility, Male/pathology* ; Pakistan ; Pedigree ; Consanguinity ; Axonemal Dyneins/genetics* ; Adult ; Spermatozoa ; Exome Sequencing

We assessed the concomitant impact of cigarette smoking and alcohol consumption in men presenting for primary couple's infertility. Data from 189 infertile men were analyzed. Semen analysis, serum hormones, and sperm DNA fragmentation (SDF) were obtained. Smoking status was categorized as follows: current nonsmoker (-S), moderate smoker (+MS), and heavy smoker (+HS). Alcohol consumption was categorized as follows: abstainer (-D), moderate drinker (+MD), and heavy drinker (+HD). Descriptive statistics and logistic regression models were applied. Among all the participants, 132 (69.8%), 30 (15.9%), and 27 (14.3%) patients were -S, +MS, and +HS, respectively. In addition, 67 (35.4%), 77 (40.7%) and 45 (23.8%) men were -D, +MD and +HD, respectively. Regarding concomitant habits, 52 (27.5%) patients were nonsmokers and abstainers (-S/-D: Group 1), 91 (48.1%) had at least one recreational habit (-S/+D or +S/-D: Group 2), and 46 (24.3%) were both smokers and drinkers (+S/+D: Group 3). Sperm concentration and progressive motility were lower in +HS and +HD, compared with -S and -D (all P < 0.05), respectively. Similarly, both parameters were significantly lower in Group 3 than Groups 1 and 2 (all P < 0.05). SDF values were higher in Group 3 than Groups 1 and 2 (both P < 0.05). In multivariate analysis, follicle-stimulating hormone (FSH) levels and concomitant +S/+D status were independent predictors of impaired sperm concentration and progressive motility (all P < 0.05). Heavy smoking and heavy drinking were associated with worse seminal parameters than moderate smoking/drinking and nonsmoking/abstaining. When concomitant, +S/+D status has an even greater detrimental effect on semen parameters.
Adult ; Alcohol Drinking/adverse effects* ; Alcoholism/complications* ; Cigarette Smoking/adverse effects* ; Cohort Studies ; Female ; Follicle Stimulating Hormone/blood* ; Humans ; Infertility, Male/pathology* ; Male ; Middle Aged ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa/ultrastructure*

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
Adult ; Caspases/metabolism* ; Cell Death ; Enzyme Activation ; Humans ; Male ; Mitochondria/pathology* ; Necrosis ; Nitrosative Stress/physiology* ; Permeability ; Peroxynitrous Acid/pharmacology* ; Phosphatidylserines/metabolism* ; Spermatozoa/ultrastructure*

The ultrastructural abnormalities of human sperm flagella can cause sperm movement disorders. Dysplasia of the fibrous sheath (DFS) is an autosomal recessive genetic disease. The affected sperm in 95-100% of the patients display short, thick and irregular tails. Transmission electron microscopy can be used to confirm the diagnosis, which reveals gross abnormal flagella, with hypertrophy and hyperplasia of the fibrous sheath, without orderly disposition in longitudinal columns and transversal ribs. The axoneme shows variable distortion or almost complete obliteration. Microtubular doublets may exhibit partial or total lack of dynein arms. The genetic etiology of DFS is not yet clear. DFS does not affect the rates of fertilization and clinical pregnancy in ICSI, but due attention should be paid to the genetic risks in the offspring of the patient.
Humans ; Hyperplasia ; complications ; pathology ; Hypertrophy ; complications ; pathology ; Infertility, Male ; Male ; Microscopy, Electron ; Sperm Motility ; physiology ; Sperm Tail ; pathology ; ultrastructure ; Spermatozoa

AIM: To investigate the histological and ultrastructural changes observed in pan masala intoxicated mammalian testes under the effect of cardamom. METHODS: Male Swiss mice were given pan masala orally at a dose of 2% of the feed and cardamom at a dose of 0.2% of the feed. They were divided into three groups, control (Group I), pan masala-treated (Group II), and a combination of pan masala and cardamom-treated group (Group III). Histologically, the testes of Group II mice displayed degeneration of tubular epithelium, disruption of spermatogenesis, and a marked reduction in germ cells. RESULTS: When cardamom was given, damage was less with fewer distorted cells and also improvement with normal tubules and spermatid differentiation in Group III. Ultrastructurally, pan masala-treated testes showed cytoplasmic vacuolation, shrinkage and pyknotic nuclei of spermatogonia, and abnormal acrosomal granules. CONCLUSION When cardamom was given, the amelioration process was more evident showing a comparable morphology with control.
Animals ; Areca ; adverse effects ; Elettaria ; Male ; Mice ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Spermatogenesis ; drug effects ; Spermatozoa ; drug effects ; Testicular Diseases ; chemically induced ; drug therapy ; pathology ; Testis ; drug effects ; pathology ; ultrastructure ; Tobacco ; adverse effects ; Tobacco, Smokeless ; adverse effects ; Vacuoles ; drug effects

OBJECTIVETo examine and analyze semen quality and sperm ultrastructural characteristics of infertile patients with varicocele.

METHODSThis study included 118 infertile patients with varicocele (the VC group) and 76 normal semen donors (the control group). We obtained routine semen parameters, seminal plasma biochemical markers and the levels of reproductive hormones in the subjects, and observed the changes in sperm structure under the scanning electron microscope and transmission electron microscope.

RESULTSCompared with the normal control, the VC patients showed significantly decreased sperm concentration, sperm progressive motility, sperm viability (P < 0.05), but no remarkable difference in semen volume and non-progressive motility (P > 0.05). The concentrations of zinc and alpha-glycoside enzyme in the seminal plasma were markedly reduced in the VC group in comparison with the controls (P < 0.05), but there was no significant difference in the level of fructose (P > 0.05), nor in such seminal plasma biochemical markers as FSH, LH, T and E2 between the two groups (P > 0.05). The percentage of morphologically normal sperm was dramatically lower in the VC than in the control group ([56.76 +/- 15.32]% vs [12.34 +/- 6.58]%, P < 0.05), and the sperm deformities were mostly in the head and neck, mainly tapering pin head accompanied by complex abnormal differentiation.

CONCLUSIONThis study demonstrated that VC may lead to oligo-astheno-terato zoospermia, and hence male infertility, which may be attributed to the changes of seminal plasma microenvironment and sperm ultrastructure.

Adult ; Case-Control Studies ; Humans ; Infertility, Male ; etiology ; pathology ; Male ; Semen Analysis ; Sperm Motility ; Spermatozoa ; ultrastructure ; Varicocele ; complications ; pathology

OBJECTIVETo study the effects of Wuzi Yanzong Pills (WYP) on sperm mitochondrial membrane potential (MMP) and its ultrastructure in oligo-asthenozoospermia model rats.

METHODSOligo-asthenozoospermia models were made in 50 male rats weighing 200 - 220 g by intragastric administration of Tripterygium Glucosides at 30 mg per kg per d for 8 weeks, and then equally allocated to a model control, a Huangjing Zanyu Capsule (HZC) control, a low-dose WYP, a medium-dose WYP, and a high-dose WYP group. Another 10 age-matched normal male rats were included as normal controls. The rats in the model and normal control groups were given intragastrically distilled water at 10 ml/kg, those in the HZC group administered HZC at 3.01 g/kg, and those in the low-, medium- and high-dose WYP groups medicated with WYP at 2.30, 4.60 and 9.20 g/kg, respectively, once daily for 30 days. At 30 minutes after the last administration, we detected the sperm MMP by JC-1 fluorescent staining and flow cytometry, and examined the sperm ultrastructure under the JEM-1230 transmission electron microscope.

RESULTSJC-1 + % and its fluorescence intensity were (33.77 +/- 6.19)% and 1 468 +/- 496 in the model control, (56.34 +/- 10.35)% and 3 277 +/- 895 in the HZC control, (40.80 +/- 10.40)% and 2 016 +/- 767 in the low-dose WYP, (59.40 +/- 6.51)% and 3 897 +/- 643 in the medium-dose WYP, and (60.71 +/- 7.81)% and 3 371 +/- 647 in the high-dose WYP group, significantly reduced in comparison with (70.80 +/- 4.92)% and 4 360 +/- 945 in the normal control group (P < 0.05), but remarkably higher in the medium- and high-dose WYP groups than in the model controls (P < 0. 05). After modeling, the sperm membrane was loose and degenerated, the mitochondria swelling, variously sized and with incomplete membrane, and the axonemal structure unclear or ruptured. After 30 days of WYP administration, compared with the model control group, the rats exhibited integrated sperm membrane and mitochondrial membrane, reduced mitochondrial swelling and basically normal axonemal and microtubular structures.

CONCLUSIONTripterygium Glucosides could decrease the sperm mitochondrial membrane potential and damage the mitochondrial structure, while WYP could significantly increase the sperm mitochondrial membrane potential and reduce the sperm mitochondrial structure damage. The protection of the integrity of sperm mitochondrial structure and function is one of the mechanisms of WYP acting on oligo-asthenozoospermia.

Animals ; Asthenozoospermia ; pathology ; physiopathology ; Drugs, Chinese Herbal ; pharmacology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Oligospermia ; pathology ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Spermatozoa ; drug effects ; physiology ; ultrastructure

OBJECTIVEGlobozoospermia is mostly associated with homozygous deletion of the DPY19L2 gene. This study aimed to investigate the DPY19L2 gene mutation in a globozoospermia patient.

METHODSWe observed the sperm histomorphology of a patient with globozoospermia using Wright-Giemsa's staining and transmission electron microscopy, detected the mutation of the DPY19L2 gene by PCR amplification and DNA sequencing, and compared the findings with the sequences issued in the Genbank.

RESULTSWright-Giemsa's staining showed that all the spermatozoa were round-headed and lacked the acrosome, with the head nucleus darkly, fully and densely stained. Transmission electron microscopy revealed larger round sperm heads, with an even layer of unit membrane surrounding the nuclei and dispersed cytoplasmic vacuoles but no acrosomal structure. No DPY19L2 gene mutation was found by PCR amplification and DNA sequencing.

CONCLUSIONNo homozygous mutation of the DPY19L2 gene was found in the globozoospermia patient, and therefore some other disease-causing genes might be involved.

Acrosome ; pathology ; ultrastructure ; DNA Mutational Analysis ; Gene Deletion ; Humans ; Infertility, Male ; genetics ; Male ; Membrane Proteins ; genetics ; Microscopy, Electron, Transmission ; Spermatozoa ; pathology ; ultrastructure