Transcriptome analysis of bull spermatozoa: implications for male fertility.

Spermatozoa deliver more than the paternal genome into the oocyte; they also carry remnant messenger RNA from spermatogenesis. The RNA profiles of spermatozoa from high-fertility and a low-fertility Holstein bulls were analysed using Affymetrix bovine genechips. A total of 415 transcripts out of approximately 24,000 were differentially detected in spermatozoa collected from both bulls (fold change > or =2.0; P<0.01). These transcripts were associated with different cellular functions and biological processes. Spermatozoa from high-fertility bulls contained higher concentrations of transcripts for membrane and extracellular space protein locations, while spermatozoa from the low-fertility bulls were deficient of transcripts for transcriptional and translational factors. Quantitative real-time PCR was used on three low-fertility and four high-fertility bulls to validate the microarray data. Two highly represented transcripts in the microarray analysis (protamine 1 and casein beta 2) were validated, as well as a third transcript (thrombospondin receptor CD36 molecule) that showed a lower concentration in low-fertility bulls. This study presents the global analysis of spermatozoa originating from bulls with opposite fertility. These results provide some specific transcripts in spermatozoa that could be associated with bull fertility.

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.

Enhanced binding of zona pellucida proteins to the acrosomal region of the intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study.

In this investigation we sought to determine whether sperm capacitation in vitro is accompanied by changes in the functional presence of zona binding sites on the plasma membrane of boar spermatozoa. During sperm incubation at 39 degrees C in various modifications of a Tyrode's-based in vitro fertilization medium, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins, using a flow cytometer. Propidium iodide was routinely included to allow simultaneous assessment of membrane integrity; rhodamine-conjugated peanut agglutinin was used to assess acrosomal status. During incubation in the fertilization medium, a subpopulation of live acrosome-intact spermatozoa developed enhanced binding of the fluorescein-conjugated solubilized zona proteins. Microscopy revealed that the increase in cytometrically detected zona binding was paralleled by an increase in the area on the sperm head to which zona proteins bound, from the apical region to the whole of the acrosomal region. The changes were accelerated by phosphodiesterase inhibitors, were attenuated by omission of bicarbonate, and were completely inhibited by addition of EGTA. In the fertilization medium, numbers of sperm showing enhanced zona binding maximized after 60-90 min. This time course is somewhat similar to that reported by others for development of egg-penetrating ability in vitro. We suggest that the observed changes in zona binding ability bring about optimal sperm-egg attachment; they may also relate to induction of the acrosome reaction by zona pellucida components. In consequence, the zona binding changes may be an important part of the process by which the sperm acquires fertilizing ability as a result of capacitation.

Identification of bovine zona pellucida glycoproteins.

Despite the economical importance of in vitro gamete technologies in cattle, only little is known about the molecular mechanisms of binding of spermatozoa to the zona pellucida (ZP) of the oocyte. The aim of the present work was to identify proteins from the bovine zona pellucida (bZP) and to investigate which bZP proteins play a role in sperm-egg binding. High resolution 2-dimensional polyacrylamide gel electrophoresis of bZP proteins under reducing conditions showed that the bovine ZP could be separated into 4 glycoprotein spots, provisionally named bZP1, bZP2, bZP3, and bZP4, with different molecular masses and isoelectrical points. The N-terminal amino acid sequence of bZP1, bZP2, and bZP4 could be determined. The N-terminal amino acid sequences of bZP1 and bZP4 were identical and were homologous to that of pZP4. Comparison of our data to that of Noguchi et al., 1994 (Biochim Biophys Acta 1201:7-14) revealed that bZP2 and bZP4 are fragments of bZP1. Immunoblot analysis showed that, respectively, anti-porcine-ZP3alpha and -ZP3beta antibodies recognized 2 distinct regions of the bZP3 spot. Both antibodies inhibited sperm-egg binding in the bovine. We conclude that the bovine ZP consists of 3 proteins that correspond by size, N-terminal amino acid sequence, and antigenic determinants of pZP1, pZP3alpha, and pZP3beta, respectively, that are encoded by the porcine ZPA, ZPB, and ZPC genes (Harris et al., 1994: J Seq Map 4:6331-393), respectively.

Induction of tissue eosinophilia by platelet-activating factor in Merino sheep.

A study was undertaken on the capacity of platelet-activating factor (PAF) to induce eosinophil accumulation in the mammary glands of non-lactating sheep. Platelet-activating factor induced dose-dependent accumulation of eosinophils in mammary exudates 24 h after infusion. Infection, by intraruminal injection of 20,000 infective Trichostrongylus colubriformis larvae, did not modify the responsiveness of outbred sheep to intramammary infusion of PAF. Mature ewes from high and low responder lines of a flock of sheep, selected on the basis of their responses to vaccination and experimental challenge with T. colubriformis as lambs, did not differ in the magnitude of the eosinophil responses to doses of PAF from 5 x 10(-13) to 5 x 10(-7) mol per gland. Intramammary infusion of an extract from third stage larvae of Haemonchus contortus elicited inflammatory exudates containing five- to ten-fold more eosinophils than that elicited by the highest dose of PAF tested. The experiments indicate that the eosinophil chemotactic agonist PAF can induce tissue eosinophilia in sheep and thus may play a role in directing the accumulation of eosinophils in tissues during disease states such as gastrointestinal parasitism.

The effect of systemic treatment with platelet-activating factor on the migration of eosinophils to lung, pleural and peritoneal cavities in the guinea pig.

Subcutaneous treatment of guinea pigs with platelet-activating factor (PAF) caused an increase in the prevalence of eosinophils in lavage fluid recovered from pulmonary airways, and pleural and peritoneal cavities. In PAF-treated animals, total numbers of eosinophils and macrophages in washings were positively correlated, thus there was no apparent competition between the two cell types for migration at traffic sites into body cavities. The results indicate that PAF acts centrally to enhance the migration of eosinophils and monocytes into body cavities, perhaps by inducing bone marrow release of both classes of leucocytes which may then migrate constitutively into lung, pleural and peritoneal cavities.