Regional differentiation in bull sperm plasma-membranes

Bull sperm heads and tails have been separated by proteolytic digestion (trypsin) and plasma membranes have been isolated, using discontinuous sucrose density gradient centrifugation. Plasma membrane bound Ca2+-ATPase is shown to be associated mostly with the tail membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization experiments indicate a more fluid lipid phase in the tail region. Differences in surface charge distribution have been found, using 1-anilinonaphthalene-8-sulfonate and Tb3+ as fluorescent probes.

Sperm chromatin in beef bulls in tropical environments

Sperm chromatin status was assessed in 565 Zebu and Zebu crossbred beef bulls in extensive tropical environments using the sperm chromatin structure assay (SCSA). The SCSA involved exposure of sperm to acid hydrolysis for 0.5 or 5.0 minutes, followed by flow cytometry to ascertain relative amounts of double-stranded (normal) and single-stranded (denatured) DNA, which was used to generate a DNA fragmentation index (%DFI). With conventional SCSA (0.5-minute SCSA), 513 bulls (91%) had <15 %DFI, 24 bulls (4%) had 15 to 27 %DFI, and 28 bulls (5%) had >27 %DFI. In 5.0-minute SCSA, 432 bulls (76%) had <15 %DFI, 68 bulls (12%) had 15 to 27 %DFI and 65 bulls (12%) had >27 %DFI. For most bulls, the SCSA was repeatable on two to four occasions; however, because most bulls had <15 %DFI, repeatability of the SCSA will need to be determined in a larger number of bulls in the 15 to 27 %DFI and >27 %DFI categories. The %DFI was negatively correlated with several bull semen parameters and the strongest negative correlation was with normal sperm. There was a strong positive correlation between %DFI and sperm head abnormalities. Based on these findings, most Zebu beef bulls in extensive tropical environments had relatively stable sperm chromatin. Based on the apparent negative correlations with conventional semen parameters, we inferred that the SCSA measured a unique feature of sperm quality, which has also been suggested for other species. Further studies on the relationships between sperm chromatin stability and fertility are required in beef bulls before chromatin status can be used as an additional predictor of the siring capacity of individual bulls in extensive multiple-sire herds. (C) 2013 Elsevier Inc. All rights reserved.

The integrity of sperm chromatin in young tropical composite bulls

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r = 0.34, P = 0.0076), Mot (r = 0.36, P = 0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r = 0.31, P = 0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r = -0.28, P = 0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r = -0.53, P < 0.0001), the percentage of sperm with head abnormalities (r = 0.68, P < 0.0001) and the percentage of intact sperm (Int) with SBH (r = -0.26, P = 0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age. (c) 2012 Elsevier Inc. All rights reserved.

Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared 'hollow' because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate-eosinophilic-clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub-adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose 'Hollow Sperm Syndrome (HSS)' to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub-adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.

Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay (SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = -0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility. © 2014 American Society of Andrology and European Academy of Andrology.

Determinants of online sperm donor success: How women choose

Because the worldwide demand for sperm donors is much higher than the actual supply available through fertility clinics, an informal online market has emerged for sperm donation. Very little empirical evidence exists, however, on this newly formed market and even less on the characteristics that lead to donor success. This article therefore explores the determinants of online sperm donors’ selection success, which leads to the production of offspring via informal donation. We find that donor age and income play a significant role in donor success as measured by the number of times selected, even though there is no requirement for ongoing paternal investment. Donors with less extroverted and lively personality traits who are more intellectual, shy and systematic are more successful in realizing offspring via informal donation. These results contribute to both the economic literature on human behaviour and on large-scale decision-making.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Action of N-bromosuccinimide on sperm whale myoglobin

Previously, it was reported from this laboratory that the heme groups of hemoglobin are “buried” within globin at pH 4.0 and not dissociated, on the basis of the obiligatory requirement of urea for the reaction of N-bromosuccinimide with the heme groups of hemoglobin at pH4.0, and also on the basis of the “normalization” of the spectrum of hemoglobin at this pH in the presence of urea or sucrose. In the present study, it has been shown that the behaviour of sperm whale myoglobin with respect to its reaction with N-bromosuccinimide and with respect to spectral “normalization” in urea or sucrose are essentially similar to that of hemoglobin. It has also been demonstrated that the spectral “normalization” obtained with crystalline hemin is not identical with that obtained with either hemoglobin or myoglobin. The bearing of the results of the present study on the earlier work on hemoglobin is indicated.

Components of fractionated stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the accessory glands before and during ejaculation. The stallion s ejaculate is a series of jets that differ in sperm concentration, semen volume and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the total number of sperm. The semen volume and sperm concentration in each of the jets decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with a low sperm concentration. The aims of these studies were to examine the effects of the different SP fractions, and the presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five experiments. The levels of selected enzymes, electrolytes and proteins in different SP fractions were determined. These studies also aimed at assessing the individual variation in the levels of the selected SP components and in the effects of SP on spermatozoa. The association between the components of SP and semen quality, sperm longevity, and fertility was examined with a stepwise linear regression analysis. Compared to samples containing SP during storage, centrifugation and the subsequent removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw motility parameters were also higher in samples containing SP compared to samples stored without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the samples stored with SP than those without SP, and this effect was seen in both sperm-rich and sperm-poor fractions. There were no differences in DNA integrity between fractions stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor fraction after SP removal. The differences between fractions in sperm motility after cooled storage were non-significant. The levels of alkaline phosphatase, acid phosphatase and β-glucuronidase were higher in the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions. The concentrations of sodium and chloride were highest in pre-sperm fluid. In the sperm-poor fraction, the level of potassium was associated with the maintenance of sperm motility during storage. The levels of alkaline and acid phosphatase were associated with sperm concentration and the total number of spermatozoa in the ejaculates. None of the measured SP components were correlated to the first cycle pregnancy rate. In summary, the removal of SP improved DNA integrity after cooled storage compared with samples containing SP. There were no differences in the maintenance of sperm motility between the sperm-rich and sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was found in the sperm-rich fractions stored without SP. In practice, the results presented in this thesis support the use of individual modifications of semen processing techniques for cooled transported semen from subfertile stallions.

Impaired spermatogenesis in Finnish boars and bulls

Maintenance of breeding efficiency and high semen quality is essential for reproductive success in farm animals. Early recognition of possible inheritable factors causing infertility requires constant attention. This thesis focuses on describing different manifestations of impaired spermatogenesis, their impact on fertility and partly also their incidence in populations. The reasons for spermatogenic failure are various. An interruption of germ cell differentiation, spermatogenic arrest, can lead to infertility. The incidence of azoospermia was investigated in the 1996 2005 survey of Finnish AI and farm breeding boars. We focused on the diagnosis, testicular morphometry and the possible reasons for the condition. The incidence of azoospermia was significantly higher in Yorkshire boars than in the Landrace breed. The most common diagnosis in Yorkshire boars was germ cell arrest at the primary spermatocyte level. The second most frequent diagnosis in Yorkshire boars was segmental aplasia of the Wolffian ducts with idiopathic epididymal obstruction. Other reasons for azoospermia were infrequent. In the second study we investigated the incidence of two relatively well-defined specific sperm defects in Finnish Yorkshire and Landrace boars during the same survey, the immotile short-tail sperm (ISTS) defect and the knobbed acrosome (KA) defect. In the Finnish Yorkshire boars the inherited ISTS defect, and the probably inherited KA defect, were important causes of infertility during 1996 2005. The ISTS defect was found in 7.6% and the KA defect in 0.8% of the Yorkshire boars. No Landrace boars were diagnosed with either of these two defects. In the third study we described a new sterilizing sperm defect in an oligoasthenoterazoospermic bull. Because of its morphological characteristics this defect was termed the multinuclear-multiflagellar sperm (MNMFS) defect. The number of Sertoli cells in the seminiferous tubuli was highly increased in the MNMFS bull compared with the number in normal bulls. In the following two studies we used a combined approach of fluorescence in situ hybridization (FISH), flow cytometry and morphometric studies to provide information on the cytogenetic background of macrocephalic bull spermatozoa. We described cellular features of diploid spermatozoa and compared the failures in the first and second meiotic divisions. In the last study we describe how the transplantation of testicular cells was used to determine whether spermatogonia derived from donor animals are able to colonize and produce motile spermatozoa in immune-competent unrelated boars suffering the ISTS defect. Transplantation resulted in complete focal spermatogenesis, indicated by the appearance of motile spermatozoa and confirmed by genotyping.

Effect of altering endogenous gonadotrophin concentrations on the kinetics of testicular germ cell turnover in the bonnet monkey (Macaca radiata)

The role of FSH and diurnal testosterone rhythms in specific germ cell transformations during spermatogenesis were investigated using DNA flow cytometry and morphometry of the seminiferous epithelium of the adult male bonnet monkey (Macaca radiata), the endogenous hormone levels of which were altered by two different protocols. (1) Active immunization of five monkeys for 290 days using ovine FSH adsorbed on Alhydrogel resulted in the neutralization of endogenous FSH, leaving the LH and diurnal testosterone rhythms normal. (2) Desensitization of the pituitary gonadotrophs of ten monkeys by chronically infusing gonadotrophin-releasing hormone analogue, buserelin (50 micrograms/day release rate), via an Alzet pump implant (s.c.) led to a 60-80% reduction in LH and FSH as well as total abolition of testosterone rhythms. The basal testosterone level (3.3 +/- 2.0 micrograms/l), however, was maintained in this group by way of an s.c. testosterone silicone elastomer implant. Both of the treatments caused significant (P < 0.01) nearly identical reduction in testicular biopsy scores, mitotic indices and daily sperm production rates compared with respective controls. The germ cell DNA flow cytometric profiles of the two treatment groups, however, were fundamentally different from each other. The pituitary-desensitized group exhibited a significant (P < 0.001) increase in 2C (spermatogonial) and decrease in 1C (round spermatid) populations while S-phase (preleptotene spermatocytes) and 4C (primary spermatocytes) populations were normal, indicating an arrest in meiosis caused presumably by the lack of increment in nocturnal serum testosterone. In contrast, in the FSH-immunized group, at day 80 when the FSH deprivation was total, the primary block appeared to be at the conversion of spermatogonia (2C) to cells in S-phase and primary spermatocytes (4C reduced by > 90%). In addition, at this time, although the round spermatid (1C) population was reduced by 65% (P < 0.01) the elongate spermatid (HC) population showed an increase of 52% (P < 0.05). This, taken together with the fact that sperm output in the ejaculate is reduced by 80%, suggests a blockade in spermiogenesis and spermiation. Administration of booster injections of oFSH at time-points at which the antibody titre was markedly low (at days 84 and 180) resulted in a transient resurgence in spermatogenesis (at day 180 and 228), and this again was blocked by day 290 when the FSH antibody titre increased.

Correlation of seasonal changes in sperm output with endocrinological changes in the adult male bonnet monkey, Macaca radiata

We have examined the monthly variations in sperm output and attempted to correlate the profiles of endocrine hormones secreted with the sperm counts throughout the ,year in the adult male bonnet monkey. As previously reported, there was a distinct spurt in sperm output beginning September through December months. A concomitant increase in serum testosterone and prolactin concentrations were also noted during September through November (mid and post-monsoon season). Although there was a marked increase in gonadotropin releasing hormone stimulated testosterone secretion, the peak testosterone concentrations post gonadotropin releasing hormone injection did not vary significantly (P>0.05) throughout the year. Basal serum follicle stimulating hormone concentrations did not vary significantly (P>0.05) during April to June months compared to September-November months. Serum inhibin concentration remained unaltered throughout the year, except in the month of March. The results of this study provide evidence for annual rhythms in prolactin and testosterone secretion and a distinct seasonality in the sperm output of the adult male bonnet monkey, but the pituitary responsiveness to exogenous gonadotropin releasing hormone remains unaltered throughout the year. Because of the existence of seasonality as noted in the present study, future studies which utilize the adult male bonnet monkey as an experimental model need to take into consideration the seasonal effects on reproductive function in this species.

Pentoxifylline improves sperm capacitation and in vitro fertilization of oocytes in the golden hamster

Pentoxifylline (PF) is used to improve motility of spermatozoa from subfertile or nonfertile males to accomplish in vitro fertilization in humans. The possible adverse effect of PF on pre- and peri- implantation stage embryo development in a suitable rodent model, such as the golden hamster, is yet to be determined. In this study, hamster cauda epididymal spermatozoa were exposed to different concentrations (0.23 to 3.6 mM) of PF, and their quantitative [percentage of motility] and qualitative [Score 0 to 5] motility were assessed and values expressed as the sperm motility index. Upon addition of spermatozoa to dishes containing PF, an immediate increase in sperm motility and sperm motility index was evident, which increased up to 4 to 6 h and then declined. The sperm motility index increase by PF was dose-dependant, and greater than or equal to 1.8 mM PF was detrimental after 4 h. The optimum dose of PF was found to be 0.45 mM. To assess the fertilizing ability of PF-treated spermatozoa, in vitro fertilization was carried out. Fertilization rates for spermatozoa treated with 3.6 mM PF were lower (53.8 +/- 7.8) than for the controls (69.5 +/- 10.2), whereas, treatment with 0.45 mM PF increased the rates (91.6 +/- 4.3) compared with that of the controls (80.2 +/- 5.9). In conclusion, low concentrations (0.23 to 0.45 mM) of PF improve sperm capacitation and fertilization of oocytes in vitro in the golden hamster.

Susceptibility of sperm chromatin to acid denaturation in situ: A study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata)

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.