Relationship between semen characteristics and fertility in electroejaculated mice

Ejaculates were obtained from C57BL mice by applying two successive series of electrical stimuli which were delivered via a bipolar rectal probe. The ejaculates thus collected contained fertile spermatozoa as indicated by results from in-vitro fertilization. Once separated from the seminal plasma, ejaculated spermatozoa possessed the same in-vitro fertilization rate as epididymal sperm. Ejaculates were analysed for coagulum weight, ejaculate volume, sperm count, sperm motility, acid phosphatase content and fructose content. Significant differences were present between several of these values for fertile and infertile mice, and values were therefore empirically assigned to represent minimal amounts for 'normal' fertility (1.5 microliters ejaculate volume; 10.2 mg coagulum weight; 2.5 x 10(6) spermatozoa/ml; 2.3 x 10(3) motile spermatozoa/ejaculate). One half of the fertile animals had no deficiencies in any of the characteristics measured, whereas 97% of the infertile animals had at least one deficiency. No fertile male had more than 2 deficiencies. These data show that the characteristics of mouse semen obtained by the present method of electroejaculation are related to the fertility status of the animal.     

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.     

鸟类婚配制度的生态学分类

在Ｅmlen和Ｏring鸟类婚配制度生态学分类系统的基础上,根据近年来鸟类行为生态学研究的成果,对鸟类的婚配制度进行了补充分类,并强调了应以进化稳定策略的观念来认识乌类的婚配制度。补充的鸟类婚配制度生态型包括：合作型一雄一雌制（cooperative monogamy)、临界型一雄一雌制（critical monogamy)、保卫雌性型一雄一雌制（female defense mognogamy)、从领域型一雄多雌制（poly-terri-tory polygyny)和社群繁殖制。合作型一雄一雌制的鸟 类雌雄个体爱力合作才保证繁殖的成功;临界型一悲欢离合一雌制鸟类雌雄个体都有多配倾向,但迫于生态压力必须共同抚育后代才能繁殖成功;保卫雌性型一雄一雌制的鸟 类通过保卫一个雌鸟不被其它雄鸟占有而保证繁殖成功,多领域型一雄多雌制的雄鸟通过占有多个领域而多个雌鸟交配;社群繁殖制的鸟 类由三个以上个体参与工部分参与繁殖,所有个体共同抚育一批后代,现有的鸟类婚配制度可以归为一雄一雌制（monogamy)、一雄多雌繁殖,所有个体共同抚育一批后代。现有的鸟类婚配制度可以归为一雄一雌制(polygyny)、一雌多雄制（polyandry)快速多窝型多配制（rapid-multiple-clutch polygamy)和社群繁殖制（social breeing systen）五大类型。     

Zona pellucida binding and zona-induced acrosome reactions in horse spermatozoa: comparisons between fertile and subfertile stallions

Diagnostic tests that probe sperm function are needed to determine the potential etiologies of subfertility and to explore treatments of subfertility in stallions. Using epifluorescence and phase contrast microscopy, a comparison was made between ejaculates from 3 fertile and 3 subfertile stallions in which sperm-zona pellucida binding and acrosomal status were measured. Motile spermatozoa were selected by Percoll gradient centrifugation and were capacitated in vitro using TEST:TALP capacitation medium at 39 degrees C under humidified air containing 5% CO2. Concentration of motile spermatozoa was held constant during co-incubation with oocytes for fertile and subfertile ejaculates. The total number of zona pellucida-bound spermatozoa was higher for fertile stallions than for subfertile stallions (P < 0.05). Similarly, the percentage of acrosome reactions in zona pellucida-bound spermatozoa was higher for the 3 fertile stallions than for the 3 subfertile stallions (P < 0.05). These results indicate that spermatozoa from fertile stallions may interact with female gametes differently from that of subfertile stallions and suggest that sperm functions are measurable and may vary with fertility.     

Sperm centriole assessment identifies male factor infertility in couples with unexplained infertility – a pilot study

Unexplained infertility affects about one-third of infertile couples and is defined as the failure to identify the cause of infertility despite extensive evaluation of the male and female partners. Therefore, there is a need for a multiparametric approach to study sperm function. Recently, we developed a Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) assay to determine sperm centriole quality. Here, we perform a pilot study of sperm from 10 fertile men and 10 men in couples with unexplained infertility, using three centriolar biomarkers measured at three sperm locations from two sperm fractions, representing high and low sperm quality. We found that FRAC can identify men from couples with unexplained infertility as the likely source of infertility. Higher quality fractions from 10 fertile individuals were the reference population. All 180 studied FRAC values in the 10 fertile individuals fell within the reference population range. Eleven of the 180 studied FRAC values in the 10 infertile patients were outliers beyond the 95% confidence intervals (P = 0.0008). Three men with unexplained infertility had outlier FRAC values in their higher quality sperm fraction, while four had outlier FRAC values in their lower quality sperm fraction (3/10 and 4/10, P = 0.060 and P = 0.025, respectively), suggesting that these four individuals are infertile due, in part, to centriolar defects. We propose that a larger scale study should be performed to determine the ability of FRAC to identify male factor infertility and its potential contribution to sperm multiparametric analysis.     

Infertile matings and sperm competition: the effect of "nonsperm representation" on intraspecific variation in sperm precedence patterns

In theoretical and experimental approaches to the study of sperm competition, it is often assumed that ejaculates always contain enough sperm of good quality and that they are successfully transferred and used for fertilization. However, this view neglects the potential effects of infertility and sperm limitation. Permanent or temporal male infertility due to male sterility, insemination failures, or failures to fertilize the ova implies that some males do not achieve sperm representation in the female reproductive tract after mating. A review of the literature suggests that rates of nonsperm representation may be high; values for the proportion of infertile matings across 30 insect species vary between 0% and 63%, with the median being 22%. I simulated P2 (the proportion of offspring fathered by the second male to copulate with a female in a double-mating trial) distributions under a mechanism of random sperm mixing when sample sizes and rates of male infertility varied. The results show that nonsperm representation can be responsible for high intraspecific variance in sperm precedence patterns and that it can generate misleading interpretations about the mechanism of sperm competition. Nonsperm representation might be a common obstacle in the studies of sperm competition and postcopulatory female choice.     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.     

Blood Serum and Seminal Plasma Selenium,Total Antioxidant Capacity and Coenzyme Q10 Levels in Relation to Semen Parameters in Men with Idiopathic Infertility

In this case–control study, we aimed to evaluate the serum and seminal plasma levels of Selenium (Se), total antioxidant capacity (TAC), and Coenzyme Q10 (CoQ-10) and determine their relationship with sperm concentration, motility, and morphology in men with idiopathic infertility. A total of 59 subjects were enrolled in the study. Forty four patients were diagnosed with idiopathic male infertility and had abnormal sperm parameters, and 15 subjects had normal sperm parameters with proven fertility. Serum Se, semen Se, and semen TAC levels were significantly different in the fertile and infertile groups (p?<?0.01, p?<?0.001, and p?<?0.001, respectively). However, serum TAC, serum, and seminal plasma CoQ-10 levels did not differ between fertile and infertile groups. When the levels of the measured parameters were compared in serum and seminal plasma, serum levels of Se were found to be correlated positively with the semen levels in all subjects included into the study (N?=?59) (r?=?0.46, p?<?0.01). A relationship was found between neither serum and semen levels of TAC nor between serum and semen levels of CoQ-10. Correlations among measured serum and semen parameters with sperm parameters demonstrated that both the serum and semen levels of Se were correlated positively with spermatozoa concentration, motility, and morphology. Additionally, seminal plasma levels of TAC correlated positively with all these sperm parameters. On the other hand, seminal plasma levels of CoQ-10 correlated only with sperm morphology but not with concentration or motility. No relationship was observed between serum levels of TAC or serum levels of CoQ-10 and sperm parameters. In conclusion, serum and seminal plasma Se deficiency may be a prominent determinant of abnormal sperm parameters and idiopathic male infertility. Measurement of serum Se levels may help determine nutritional status and antioxidant capacity in infertile patients, which may help distinguish those patients who will benefit from supplementation therapy.     

Morphological abnormalities in the spermatozoa of fertile and infertile men

The morphological analysis of the spermatozoa from fertile and infertile men was performed using light and electron microscopy to clarify the relationship between sperm morphology and fertility. Semen samples obtained from 22 partners of pregnant women were prepared according to the protocol standardized in an international collaborative study. Semen samples from 17 patients with asthenozoospermia or varicocele were collected in a hospital. Abnormalities in the spermatozoa were classified into three types for the tails, two for the midpieces, and six for the heads according to the criteria adapted from WHO guidelines (World Health Organization, 1999: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction (4th edition)). Approximately 14% of the spermatozoa from the fertile men had abnormal tails at the light microscopic level while approximately 44% had abnormal heads. Most types of abnormalities found in the spermatozoa from the asthenozoospermic and varicocele patients were encountered in those from the fertile men, although the semen from the fertile men contained a higher percentage of normal spermatozoa than that from the patients. These results were also confirmed at the ultrastructural level. Most abnormal cell types are encountered in semen from fertile men, although the incidence of abnormalities is low.     

Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) with single-color chromosome-specific probes was used to study the rates of disomy for chromosome 1, 16, X, and Y in sperm of fertile and infertile subjects. Diploidy rates were studied using a two-color cocktail of probes for chromosomes 17 and 18 in the same sperm samples. Two-color methodology was not available at the outset of the study. A total of 450,580 spermatozoa were studied from 21 subjects (9 fertile, 12 infertile). Significant differences were observed in the disomy rates between chromosomes with the highest frequency observed for chromosome 16 (0.17%) and the lowest for the Y chromosome (0.10%). No differences were observed between fertile and infertile subjects for either diploidy or disomy. Total disomy rates for chromosomes 1, 16, X and Y ranged from 0.34% to 0.84% among infertile subjects, and 0.32% to 0.61% among fertile subjects. Our data suggest that generalized aneuploidy in sperm is not a major contributor to unexplained infertility.     

Observations on egg production by Toxocara pteropodis

Studies in juvenile Pteropus poliocephalus showed an average daily egg production by Toxocara pteropodis of 25,000 per female, with concentrations of up to 16,000 epg. regardless of whether eggs were fertile or infertile. Production commenced as early as 35 and as late as 48 days post-partum and rose to plateau average levels over about 10 days. For 23 days one bat passed infertile eggs which, over 2 days, were then replaced completely by fertile eggs. The implicit delay in maturation of a male nematode suggests that transmammary passage of larvae to suckling bats may persist for at least 3 weeks. Patency was terminated by the spontaneous expulsion of worms. If male worms were lost first, the egg output converted from fertile to 100% infertile within 48 h and the females were devoid of spermatozoa, suggesting that T. pteropodis copulate at least once daily. In prolonged infections, worm fecundity and egg fertility diminished, so that females with stored spermatozoa were producing mixtures of fertile and infertile eggs.     

Genetic conflicts, intrinsic male fertility, and ejaculate investment

Few aspects of biology are linked to so many evolutionary conflicts as sperm production and fertilization. Segregation distortion and maternal inheritance of cytoplasmic genes, causing maladapted males, are common sources of variation in the competitive ability of sperm, leading males to vary in their intrinsic fertility. Here, I theoretically analyze the effect of such variation in male intrinsic fertility on ejaculate investment. The model reveals that with increasing variation in male fertility, males should overall spend less resources on their ejaculates. Furthermore, if males differing in intrinsic fertility are able to invest differently in sperm production, there are two contrasting outcomes. Typically, less fertile males should invest more. However, if female mating frequency is relatively low and differences between males relatively large, the most common male genotype should invest more. These results have important consequences both for the understanding of sperm competition strategies as well as for the evolution of female polyandry and female mating preferences.     

Immunodominant semen proteins I: New patterns of sperm proteins related to female immune infertility

Infertility affects approximately 10% of couples at reproductive age. Semen constituents may be potential immunogenic structures for women. The aim of our work is to detect and identify sperm proteins interacting with serum IgG antibodies from women with fertility disorders. The biochemical characterization of sperm antigens was performed using one and two dimensional gel electrophoresis, both of which were followed by immunoblotting. The IgG-binding proteins of interest were identified using mass spectrometry. From the serum pool of 30 infertile women, we detected sperm antigens within a relative molecular mass range between 30–80 kDa with an isoelectric point of 4–7. No antigens were detected using the serum pool from 10 fertile women (control group). Heat shock proteins (HSP 70) were identified as major sperm antigens associated with female immune infertility. Additionally, we report for the first time that alpha-enolase is a significant sperm antigen from the serum pool of infertile women. We suggest that the IgG-binding proteins identified in our study are related to immune infertility in the case of certain women with abnormally high levels of IgG antibodies linked to sperm proteins. Our results might be useful in the diagnoses of female immune infertility and may provide potential targets for further therapeutic treatment.     

Preservation of ejaculated mouse spermatozoa from fertile C57BL/6 and infertile Hook1/Hook1 mice collected from the uteri of mated females

Methods routinely used to preserve mouse spermatozoa require that the male be killed to recover spermatozoa from the epididymides. Here we obtained multiple samples of ejaculated spermatozoa from normal fertile C57BL/6 and infertile Hook1/Hook1 (formerly known as azh/azh) mutant males from uteri after mating, thus avoiding termination of the males. Ejaculated sperm were preserved by conventional cryopreservation or by rapid freezing without cryoprotection, and were injected into the oocytes by intracytoplasmic sperm injection (ICSI). The proportions of oocytes that survived, became activated, and developed into two-cell embryos were similar when comparing the two preservation methods in wild-type versus Hook1/Hook1 mice and tested mice versus controls (fresh and rapid-frozen epididymal and fresh ejaculated sperm). Two-cell embryos were transferred into the oviducts of pseudopregnant females, and fetal development was examined at Day 15 of gestation. A total of 39%-54% of transferred embryos produced with preserved ejaculated sperm implanted. Live, normal fetuses (11%-17%) were obtained in all examined groups and from all males included in the study. More implants (71%-82%) and fetuses (28%-31%) were noted in controls. Lower developmental potentials of embryos produced with preserved ejaculated sperm might be due to their capacitation status; the majority of sperm retrieved from the uterus were capacitated. This study bears significance for the maintenance and distribution of novel mouse strains. The method is applicable for all types of mice, including those with male infertility syndromes. The sole requirement is that the male of interest is able to copulate and its ejaculate contains spermatozoa.     

Mutations in a novel locus on mouse chromosome 11 resulting in male infertility associated with defects in microtubule assembly and sperm tail function

Traditional gene knock-out approaches using homologous recombination in embryonic stem cells are routinely used to provide functional information about genes involved in reproduction. In the present study, we examined a novel approach using N-ethyl-N-nitrosourea (ENU) together with a balancer chromosome mating strategy to identify new loci with functional roles in male fertility. Our genetic strategy is a forward-genetic approach; thus, our phenotypic investigation begins with the discovery of an abnormal phenotype without previous knowledge of the mutant locus. We isolated eight recessive mutations on chromosome 11 that resulted in male or female infertility from a screen of 184 founder pedigrees from ENU-treated males. After testing the six male infertile and two female infertile mutations for their ability to complement, we found that three independent recessive male infertile mutations failed to complement each other. The male infertility was associated with reduced epididymal sperm count, a block in late-spermatid differentiation, and increased apoptosis. Furthermore, the three male infertile mutants had severe defects in epididymal sperm morphology associated with incorrect microtubule assembly. Electron microscopy revealed unique defects in sperm head and tail morphology for each of the three alleles. One allele had an abnormal manchette assembly of the sperm head. The other two alleles had different abnormalities in the 9+2 patterning of the microtubules in the sperm tail axoneme, with one containing only five of the microtubule doublets and the other containing an extra doublet. The isolation of this allelic series identifies a new locus on mouse chromosome 11 that is required for spermiogenesis and male fertility.     

Effect of trace elements on the seminal oxidative status and correlation to sperm motility in infertile Saudi males

The impact of trace elements, especially zinc, selenium, copper, and magnesium, on male fertility has gained great interest and significance. Increased oxidative stress and altered trace element levels are probable etiological factors underlying male reproductive dysfunction and infertility. The present study focused on the evaluation of seminal oxidative markers, such as reactive oxygen species (ROS), malondialdehyde (MDA), and total antioxidant capacity (TAC), and trace element levels in the normozoospermic fertile control group (n = 40) and asthenozoospermic infertile group (n = 30). Semen from infertile men exhibited significantly higher ROS and MDA levels accompanied with significant decline in TAC and trace element (zinc and magnesium) levels. Furthermore, a significant correlation was observed between trace elements and oxidative markers with sperm motility. The current study revealed increased lipid peroxidation and oxidant-reductant imbalance that leads to deterioration of semen quality and male infertility. Thus, oxidative stress and trace elements can be considered important biomarkers of male infertility. Measurement of seminal oxidative stress with conventional seminological parameters must be integrated in fertility assessment from early stages to ensure healthy semen characteristics and fertility in men.     

Cadmium effects on sperm morphology and semenogelin with relates to increased ROS in infertile smokers: An in vitro and in silico approach

Smoking releases cadmium (Cd), the metal toxicant which causes an imbalance in reactive oxygen species level in seminal plasma. This imbalance is envisaged to impair the sperm DNA morphology and thereby result in male infertility. In order to correlate this association, we performed in vitro and in silico studies and evaluated the influence of reactive oxygen species imbalance on sperm morphology impairments due to smoking. The study included 76 infertile smokers, 72 infertile non-smokers, 68 fertile smokers and 74 fertile non-smokers (control). Semen samples were collected at regular intervals from all the subjects. Semen parameters were examined by computer assisted semen analysis, quantification of metal toxicant by atomic absorption spectrophotometer, assessment of antioxidants through enzymatic and non-enzymatic methods, diagnosis of reactive oxygen species by nitro blue tetrazolium method and Cd influence on sperm protein by in vitro and in silico methods. Our analysis revealed that the levels of cigarette toxicants in semen were high, accompanied by low levels of antioxidants in seminal plasma of infertile smoker subjects. In addition the investigation of Cd treated sperm cells through scanning electronic microscope showed the mid piece damage of spermatozoa. The dispersive X-ray analysis to identify the elemental composition further confirmed the presence of Cd. Finally, the in-silico analysis on semenogelin sequences revealed the D-H-D motif which represents a favourable binding site for Cd coordination. Our findings clearly indicated the influence of Cd on reactive oxygen species leading to impaired sperm morphology leading to male infertility.     

Is there a relationship between sperm chromosome abnormalities and sperm morphology?

This review explores the relationship between sperm chromosomal constitution and morphology. With the advent of techniques for obtaining information on the chromosome complements of spermatozoa, this relationship has been studied in fertile men and in men with a high frequency of chromosomal abnormalities. Using human sperm karyotype analysis, no relationship between sperm chromosome abnormalities and morphology was found in fertile men, translocation carriers or post-radiotherapy cancer patients. Fluorescence in situ hybridization (FISH) analysis has not generally revealed a specific association between morphologically abnormal sperm and sperm chromosome abnormalities, but has indicated that teratozoospermia, like other forms of abnormal semen profiles (aesthenozoospermia, oligozoospermia) is associated with a modest increase in the frequency of sperm chromosome abnormalities. However, FISH studies on some infertile men and mouse strains have suggested that certain types of morphologically abnormal spermatozoa, such as macrocephalic multitailed spermatozoa, are associated with a very significantly increased frequency of aneuploidy. Thus, there may be an association between sperm morphology and aneuploidy in infertile men with specific abnormalities.