Timing of sperm capacitation varies reproducibly among men

Sperm must mature functionally in the process of capacitation to become able to fertilize. Capacitation depends on membrane lipid changes, and can be quantitatively assessed by redistribution of the ganglioside G_M1, the basis of the Cap‐Score? sperm function test. Here, differences in Cap‐Score were compared among and within men at two time points. Ejaculates were liquefied, washed, and incubated for 3 hr under capacitating (Cap) conditions, then fixed and analyzed immediately (Day0); after being incubated 3 hr under Cap conditions then maintained 22–24 hr in fix (Day1‐fix); or after 22–24 hr incubation under Cap conditions prior to fixation (Day1). In all cases, a light fixative previously shown to allow membrane lipid movements was used. Day1‐fix and Day1 Cap‐Scores were greater than Day0 (p < 0.001; n = 25), whereas Day1‐fix and Day1 Cap‐Scores were equivalent (p = 0.43; n = 25). In 123 samples from 52 fertile men, Cap‐Score increased more than 1SD (7.7; calculated previously from a fertile cohort) from Day0 to Day1‐fix in 44% (54/123) of the samples. To test whether timing of capacitation was consistent within an individual, 52 samples from 11 fertile men were classified into either “early” or “late” capacitation groups. The average capacitation group concordance within a donor was 81%. Median absolute deviation (MAD; in Cap‐Score units) was used to assess the tightness of clustering of the difference from Day0 to Day1‐fix within individuals. The average (2.21) and median (1.98) MAD confirmed consistency within individuals. Together, these data show that the timing of capacitation differed among men and was consistent within men.

     

Validation of a laboratory‐developed test of human sperm capacitation

Sperm must undergo capacitation to become fertilization competent. Here we validated that monosialotetrahexosylganglioside (G_M1) localization patterns, which were assessed in the Cap‐Score? Sperm Function Test, reflect a capacitated state in human sperm. First, we defined patterns representing sperm that do or do not respond to stimuli for capacitation. Sperm with “capacitated” patterns had exposed acrosomal carbohydrates and underwent acrosome exocytosis in response to calcium ionophore (A23187). Precision was evaluated by percent change of the Cap‐Score measured for 50, 100, 150, and 200 sperm. Changes of 11%, 6%, and 5% were observed (n ≥ 23); therefore, we counted ≥150 sperm per condition. Variance within and between readers was evaluated using 20 stitched image files generated from unique ejaculates. Two trained readers randomly resampled each image 20 times, reporting an average standard deviation of 3 Cap‐Score units and coefficient of variation of 13% when rescoring samples, with no difference between readers. Semen liquefaction times ≤2 hr and mechanical liquefaction with Pasteur or wide‐orifice transfer pipettes did not alter Cap‐Score values. However, liquefaction with chymotrypsin (p = 0.002) and bromelain (p = 0.049) reduced response to capacitating stimuli and induced membrane damage, while counterintuitively improving sperm motility. Together, these data validate the Cap‐Score assay for the intended purpose of providing information on sperm capacitation and male fertility. In addition to its clinical utility as a diagnostic tool, this test of sperm function can reveal the impact of common practices of semen handling on the ability of sperm to respond to capacitation stimuli.

     

Expectations of assisted conception for infertility.

OBJECTIVE--To provide reliable prognostic information for couples seeking assisted conception. DESIGN--Analysis of four years'' practice (1988-91). SETTING--Private university service linked with NHS reproductive medicine services. PATIENTS--804 couples with various causes of subfertility, median duration five years, median age of women 34 years. INTERVENTIONS--1280 completed cycles: 950 in vitro fertilisation, 144 gamete intrafallopian transfer, and 186 intrauterine insemination and superovulation. MAIN OUTCOME MEASURES--Pregnancy and birth rates per cycle and cumulative pregnancy and take home baby rates per couple. RESULTS--In women under 40 years and men with normal sperm, whatever the cause of infertility, results with in vitro fertilisation improved steadily reaching a pregnancy rate per cycle of 30% (95% confidence interval 26% to 35%) during 1990-1 and birth rate per cycle of 29% (23% to 35%) in 1990. Pregnancy and birth rates for gamete intrafallopian transfer were 36% (28% to 44%) and 26% (17% to 37%) and for intrauterine insemination 18% (12% to 24%) and 16% (10% to 22%). After six cycles cumulative probability of pregnancy was 82% and cumulative take home baby rate 70%. Considering only in vitro fertilisation and gamete intrafallopian transfer after four cycles the pregnancy rate was 78% (66% to 91%). CONCLUSIONS--Conception is less likely in women over 40 and men with sperm dysfunction. For other couples the prognosis for a live birth is at least as good as for fertile couples if they persist with treatment.     

Differences in the Endocannabinoid System of Sperm from Fertile and Infertile Men

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB₁, CB₂) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB₁, CB₂ and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB₁ and CB₂ receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.     

Stratégies biologiques à l’heure de l’Assistance Médicale à la Procréation chez le blessé médullaire

Most men with spinal cord injury (SCI) men have fertility problems caused by anejaculation and decreased fertility of the ejaculate. There are two main causes for the impaired reproductive potential in SCI men: ejaculatory dysfunction and poor quality semen. However, current treatment techniques allow a large number of SCI males to achieve ejaculation (rectal electro-stimulation, penile vibrator stimulation). Firstly, masturbation and/or penile vibrator are used at home allowing the couple to perform insemination themselves. The semen of men with spinal cord injuries has commonly been characterised by small volume, abnormal count (low or high), decreased sperm mobility, increased formation of reactive oxygen species, sperm autoimmunity, necrospermia. This impairment is thought to be due to insufficient drainage, genitourinary infections and raised scrotal temperature. Testicular biopsy reveals varying degrees of tubule degeneration and decreased spermatogenetic activity. Semen could be used for various assisted reproductive technologies such as intrauterine insemination,in vitro fertilisation (IVF) and microinsemination (ICSI). The literature reports pregnancy rates by intrauterine insemination of about 15 to 20% per couple. Clinical pregnancy rates after IVF and ICSI techniques are 30% per cycle and these results are comparable to the clinical pregnancy rates when these techniques are performed for female infertility. However, semen must be frozen as soon as possible after the injury and the patient must be informed about the various available assisted reproductive technologies.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

Aging and Loss of Fertility in Male and Female Brachionus plicatilis (Rotifera)

Summary

The decline with age of mictic female susceptibility to fertilization and male capacity for fertilization is characterized for the rotifer Brachionus plicatilis. All mictic females were susceptible to fertilization until age 4 hr. Susceptibility then declined non-linearly according to the quadratic equation Y = 140.6—14.3X + 0.36X ². By age 24 hr, sexual females no longer could be fertilized. Only 83% of newborn males were capable of fertilization. This level of fertility held until age 8 hr, then declined linearly. The age when 50% of individuals were no longer fertile was termed the length of fertilizability ₅₀(LF₅₀) and is 7.9 hr (16.7% of lifespan) and 18.8 hr (26.1% of lifespan) for females and males, respectively. Newborn males had an average of 30.1 ± 1.40 motile sperm. Males transferred a mean of 2.3 motile sperm into the pseudocoelom of females on each insemination. Sperm inseminated per copulation closely corresponds to the mean number of resting cysts produced by fertilized females. It is not likely that resting cyst production is limited by sperm availability.     

Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide

There is an urgent need to develop a better method of contraception which is non‐steroidal and reversible to control world population explosion and unintended pregnancies. Contraceptive vaccines (CV), especially targeting sperm‐specific proteins, can provide an ideal contraceptive modality. Sperm‐specific proteins can induce an immune response in women as well as men, thus can be used for CV development in both sexes. In this article, we will review two sperm‐specific proteins, namely Izumo protein and YLP₁₂ dodecamer peptide. Gene‐knockout studies indicate that Izumo protein is essential for sperm–egg membrane fusion. Vaccination with Izumo protein or its cDNA causes a significant reduction in fertility of female mice. The antibodies to human Izumo inhibit human sperm penetration assay. Recently, our laboratory found that a significant percentage of infertile women have antibodies to Izumo protein. The second sperm‐specific protein is YLP₁₂, a peptide mimetic sequence present on human sperm involved in recognition and binding to the human oocyte zona pellucida. Vaccination with YLP₁₂ or its cDNA causes long‐term, reversible contraception, without side effects, in female mice. Infertile, but not fertile, men and women have antibodies to YLP₁₂ peptide. Our laboratory has isolated, cloned, and sequenced cDNA encoding human single chain variable fragment (scFv) antibody from infertile men which reacts with YLP₁₂ peptide. The human YLP₁₂ scFv antibody may provide a novel passive immunocontraceptive, the first of its kind. In conclusion, sperm‐specific Izumo protein and YLP₁₂ peptide can provide exciting candidates for antisperm CV development.     

Case series of fertility treatment in HIV-discordant couples (male positive, female negative): the Ontario experience

The success of combination antiretroviral therapies for the treatment of human immunodeficiency virus (HIV) has resulted in prolonged life expectancy (over 40 years from diagnosis) and an improved quality of life for people living with HIV. The risk of vertical HIV transmission during pregnancy has been reduced to less than 1%. As a result of these breakthroughs and as many of these individuals are of reproductive age, fertility issues are becoming increasingly important for this population. One population in which conception planning and reduction of horizontal HIV transmission warrants further research is HIV-discordant couples where the male partner is HIV-positive and the female partner is HIV-negative. Sperm washing is a technique carried out in a fertility clinic that separates HIV from the seminal fluid. Although sperm washing followed by intrauterine insemination significantly reduces the risk of horizontal HIV transmission, there has been limited access to the procedure in North America. Furthermore, little is known about the conception decision-making experiences of HIV-discordant couples who might benefit from sperm washing. Chart reviews and semi-structured interviews were completed with 12 HIV-discordant couples in Ontario, Canada. Couples were recruited through HIV clinics and one fertility clinic that offered sperm washing. Participants identified a number of factors that affected their decision-making around pregnancy planning. Access to sperm washing and other fertility services was an issue (cost, travel and few clinics). Participants identified a lack of information on the procedure (availability, safety). Sources of support (social networks, healthcare providers) were unevenly distributed, especially among those who did not disclose their HIV status to friends and family. Finally, the stigmatisation of HIV continues to have a negative affect on HIV-discordant couples and their intentions to conceive. Access to sperm washing and fertility service is significantly limited for this population and is accompanied with a number of challenges.     

A comparison of adiposity measures as predictors of all-cause mortality: the Melbourne Collaborative Cohort Study

Objective: Our goal was to examine five different measures of adiposity as predictors of all‐cause mortality. Research Methods and Procedures: Subjects were 16,969 men and 24,344 women enrolled between 1990 and 1994 in the Melbourne Collaborative Cohort Study (27 to 75 years of age). There were 2822 deaths over a median follow‐up period of 11 years. BMI, waist circumference, and waist‐to‐hip ratio were obtained from direct anthropometric measurements. Fat mass and percentage fat were estimated by bioelectric impedance analysis. Results: Comparing the top quintile with the second quintile, for men there was an increased risk of between 20% and 30% for all‐cause mortality associated with each of the anthropometric measures. For women, there was an increased risk of 30% (95% confidence interval for hazard ratio, 1.1–1.6) observed for waist circumference and 50% (1.2–1.8) for waist‐to‐hip ratio, but little or no increased risk for BMI, fat mass, and percentage fat. Waist‐to‐hip ratio was positively and monotonically associated with all‐cause mortality for both men and women. There was a linear association between waist circumference and all‐cause mortality for men, whereas a U‐shaped association was observed for women. Discussion: Measures of central adiposity were better predictors of mortality in women in the Melbourne Collaborative Cohort Study compared with measures of overall adiposity. We recommend measuring waist and hip circumferences in population studies investigating the risk of all‐cause mortality associated with obesity. The use of additional measures such as bioelectric impedance is not justified for this outcome.     

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA^®) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.     

Prise en charge de l’infécondité dans les troubles de l’éjaculation: avis conjoints de l’andrologue, du biologiste et du gynécologue

It is estimated that eighty percent of men who present ejaculatory disorders would like to procreate but only 5–10% from them can do it. Ejaculation can be achieved by assisted-techniques used in association with pharmacological treatment. In cases of failure of ejaculation and azoospermia, epididymal and testicular chirurgical sperm extraction allows obtaining spermatozoa. Less numerous than those obtained in ejaculated sperm, these chirurgical spermatozoa offer the advantage to be not infected by bacteria and by seminal plasma. The quality of sperm is evaluated by conventional spermiologic methods and it is important to analyze presence of spermatozoa in the urine together with that in ejaculated sperm. In ejaculatory disorders, the quality of sperm is often impaired and the mobility and vitality of spermatozoa are abnormal. Numerous bacteria and leucocytes are also present. These changes seem to be related to many factors located in the seminal plasma rather than central and testicular damages. As in fertile and infertile man, cryopreservation causes a decrease in conventional variables. However, taking into account the difficulty to obtain sperm, all patients with ejaculatory disorders would benefit from semen cryopreservation. In order to achieved successful pregnancy, several fertility treatment are available: home insemination with semen obtained by vibroejaculation, intrauterine insemination, in vitro fecondation, or intracytoplasmic sperm injection. Counselling couples undergoing such treatment program needs coordinated efforts of different specialities, which may involve andrology, biology and gynaecology.     

Association of Cholecystokinin 1 Receptor and β3‐Adrenergic Receptor Polymorphisms with Midlife Weight Gain

We investigated the relationship of polymorphisms in the cholecystokinin 1 receptor [CCK1R; G to T (n‐128), A to G (n‐81)] and the β₃‐adrenergic receptor (β₃‐AR; Trp64Arg) with midlife weight gain. The participants were 1012 Japanese men and women (40 to 59 years of age). Their weight at 18 years old was obtained from a questionnaire. Weight change was defined as the current weight minus the weight at 18 years old. Subjects were grouped into four categories by these genotypes: W/W = noncarriers, W/H = Arg⁶⁴ carriers of the β₃‐AR, H/W = T (n‐128) or G (n‐81) carriers of the CCK1R, H/H = T (n‐128) or G (n‐81) and Arg⁶⁴ carriers. In men, the interaction between the CCK1R and β₃‐AR polymorphisms was significant (two‐way ANOVA, p < 0.05), but neither the CCK1R nor the β₃‐AR was individually associated with weight gain. The H/H group showed a higher possibility of weight gain of 10 kg or more compared with the W/W group in men. The odds ratio for weight gain (≥10 kg) of H/H was 2.54 (95% confidence interval: 1.50 to 4.30) compared with W/W. In women, neither main effect nor interaction was significant. These results suggest that the combination of CCK1R and the β₃‐AR polymorphisms is a contributing factor for midlife weight gain in men.     

Transcervical artificial insemination in the domestic ferret (Mustela putorius furo)

The research was undertaken to develop a successful nonsurgical procedure for artificially inseminating ferrets. A fiberoptic endoscope used in conjunction with a specially designed speculum and catheter permitted cervical catheterization and intrauterine insemination. Sperm were collected from the cauda epididymides of 10 discarded breeder males; the number of sperm in diluted samples used for insemination ranged from 4.4–13.6 × 10⁶/100 μl with progressive motility of sperm ranging from 40 to 60%. Sperm collected from each male were diluted with an egg-yolk extender (TEST) and used to inseminate 8–12 females, with deposition of sperm intravaginally or transcervically into the uterine body 0 or 24 hr after an ovulatory injection of human chorionic gonadotropin (hCG). The vaginal inseminations were used as a control, and no pregnancies resulted after insemination of 26 females. Intrauterine inseminations resulted in 4/24 (17%) of the ferrets pregnant when hCG administration was coincident with insemination, and 19/24 (79%) of the ferrets were pregnant when inseminations were done 24 hr after hCG administration. All inseminated females were euthanized on day 20 after insemination to count fetuses. The mean number of fetuses was 3.1 (range, 1–8). The number (millions) of motile sperm inseminated (X) had a significant effect on the percentage of fetuses (Y). Regression analysis indicated a linear relationship between the two variables, with an R² value of 0.99 and a line of best fit described by the equation Y = 0.029 + 0.034 X. This paper is the first report of transcervical artificial insemination in the domestic ferret (Mustela putorius furo). The method can serve as a model for application to ferrets and other mammals, particularly endangered species. Zoo Biol 17:393–404, 1998. © 1998 Wiley-Liss, Inc.     

In vitro assessment of sperm from bulls of high and low field fertility

The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 °C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 °C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilize oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalize residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95% CI 39.5 to 75.3) compared to the low fertility (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95% CI 60.9 to 89.4) and 55.3 (95% CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95% CI 21.1 to 49.6) and 24.2 % (95% CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilize oocytes in vitro; however, once fertilization occurred subsequent embryo development was not significantly affected by fertility status.     

Sperm mortality, insemination and fertilization in the damselfly Ischnura senegalensis: comparisons between wild and inbred populations

Inbreeding can have deleterious effects on individual or population fitness. To avoid fitness reduction, individuals may adopt behavioral or physiological mechanisms to reduce their investment in the production of offspring with genetically similar mates. We examined whether insemination by inbred males introduced more dead sperm than insemination by wild males by counting sperm in female Ischnura senegalensis (Rambur) sperm storage organs. If inbred males inseminated fewer or lower-quality sperm, females would avoid inferior sperm. Our results revealed three features of damselfly inbreeding: insemination failed in a larger proportion of inbred pairs than in wild pairs, inbred pairs showed significantly reduced fertility, and the numbers of live and dead sperm in an inbred female’s sperm storage organs did not differ from those in wild females. These results suggested that neither sperm quantity nor sperm quality was responsible for low fertility to a significant extent, but some kind of female quality, such as sperm usage or storing ability, was. Although inbred pairs had lower fertility, there were no significant differences between inbred and wild pairs in the total numbers of live or dead sperm. It thus seemed that female choice at the insemination stage was responsible for low fertility rather than sperm quantity or quality measured by live-to-dead ratio.     

Modification of the zona-free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility

These studies were designed to evaluate the ability of the zona-free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona-free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona-free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona-free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona-free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona-free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.     

Single nucleotide polymorphisms at the adiponectin locus and risk of coronary heart disease in men and women

Objective: The objective was to examine the association of 5 common single nucleotide polymorphisms (SNPs) at the adiponectin locus with risk of coronary heart disease (CHD) in men and women. Methods and Procedures: We genotyped five common SNPs in the adiponectin gene (rs266729, ?11365C>G; rs822395, ?4034A>C; rs822396, ?3964A>G; rs2241766, +45T>G; and rs1501299, +276G>T) in men (Health Professionals Follow‐up Study) and women (Nurses’ Health Study) in a nested case control setting. Among participants free of cardiovascular disease at baseline, 266 men and 249 women developed non‐fatal myocardial infarction or fatal CHD during 6 and 8 years of follow‐up, respectively. In addition, 564 men had coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty. Using risk set sampling, controls were selected 2:1 matched on age, smoking, and date of blood draw. Results: The ?4034CC genotype was related to an increased risk of non‐fatal myocardial infarction or fatal CHD compared with the AA genotype [relative risk (RR), men, 1.69; 95% confidence interval (CI), 0.99 to 2.89; women, 2.04; 95% CI, 1.20 to 3.49); however, this genotype was not related to risk of coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty or to plasma adiponectin levels. Other SNPs or haplotypes defined by the 5 SNPs were not consistently related to risk of CHD in men and women or to plasma adiponectin levels. Discussion: Our study does not support the hypothesis that these 5 common SNPs in the adiponectin gene play an important role in the development of CHD among men and women, although we cannot exclude an association between the ?4034CC genotype and risk of CHD.     

Mechanisms underlying the micron‐scale segregation of sterols and GM1 in live mammalian sperm

We demonstrate for the first time that a stable, micron‐scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, G_M1, localizes to this same domain in live sperm. Interestingly, the G_M1 undergoes an unexplained redistribution upon cell death. We now demonstrate that G_M1 is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing G_M1 in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the “acrosome reaction.” Comparison of the dynamics of CTB‐bound endogenous G_M1 and exogenous BODIPY–G_M1 in live murine sperm demonstrate that the sub‐acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol‐ and G_M1‐enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or G_M1 that can be separated biochemically. J. Cell. Physiol. 218: 522–536, 2009. © 2008 Wiley‐Liss, Inc.