CetSINEs and AREs are not SINEs but are parts of cetartiodactyl L1

Short interspersed repetitive elements (SINEs) are widely distributed among the genomes of eukaryotes. We proposed previously that a SINE should be defined by the presence of a region homologous to a tRNA or to 7SL RNA, together with A-box and B-box promoter sequences, in order to distinguish SINEs from other short repetitive sequences, such as short segments of LINEs (long interspersed repetitive elements; Okada et al. Gene 205, 229–243, 1997). Numerous SINE sequences have been deposited to date in DNA databases. In some cases, however, designation of a particular sequence is problematic when the short repetitive sequence has been defined as a SINE without reference to the presence or absence of promoter elements specific for RNA polymerase III. We demonstrate here that four different sequences, namely, ARE1p, ARE2p, CetSINE1, and CetSINE2, each of which has been reported as a SINE, are, in fact, only partial sequences of members of a new subfamily of L1. We also demonstrate that members of this subfamily are distributed specifically among the genomes of cetartiodactyls. Received: 3 May 2000 / Accepted: 22 August 2000     

Effect of spermatozoal concentration and number on fertility of frozen equine semen

Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy rates. Several ejaculates from each of 5 stallions were frozen in a skim milk-egg yolk based freezing medium at 2 spermatozoal concentrations in 0.5-mL polyvinyl-chloride straws. Half of each ejaculate was frozen at 400 x 10(6) cells/mL and half at 1,600 x 10(6) cells/mL. Insemination doses were based on post-thaw spermatozoal motility and contained approximately 320 x 10(6) (320 to 400) motile spermatozoa or approximately 800 x 10(6) (800 to 900) motile spermatozoa. Sixty-three mares were assigned to 1 of 4 spermatozoal treatments (1--low spermatozoal number, low concentration; 2--low spermatozoal number, high concentration; 3--high spermatozoal number, low concentration; 4--high spermatozoal number, high concentration) and were inseminated daily. Post-thaw spermatozoal motility was similar for cells frozen at both spermatozoal concentrations (P > 0.1). One-cycle pregnancy rates were 15, 40, 28 and 33%, respectively, for Treatments 1, 2, 3 and 4. Packaging spermatozoa at the high concentration tended to increase pregnancy rates vs packaging at the low concentration (37 vs 22%; P = 0.095). Furthermore, when the lower spermatozoal number was used, there tended (P < 0.1) to be a higher pregnancy rate if spermatozoa were packaged at the higher concentration. There was no increase in pregnancy rates when higher numbers of motile spermatozoa were inseminated (27 vs 31%; P > 0.1). Based on these results, a single 0.5-mL straw dose containing 800 x 10(6) spermatozoa should be used and each insemination dose should contain approximately 320 x 10(6) motile spermatozoa. Fertility trials utilizing other freezing extenders are necessary before recommending a single 0.5-mL insemination dose for all freezing extenders.     

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [ ¹²⁵ I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/10 ⁶ spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.     

Two Tandemly Arrayed Transfer-RNA-Derived SINEs of the Medaka (<Emphasis Type="BoldItalic">Oryzias latipes</Emphasis>)

: Two tandemly arrayed short interspersed repetitive element (SINE) sequences were found in medaka (Oryzias latipes). These two SINE sequences, designated SINE1 and SINE2, were flanked by a 180-bp AT-rich region. Both appeared to be derived from transfer RNA. The former exhibited 80% sequence homology to human tRNA^Ala and the latter exhibited 94% sequence homology to rat tRNA^Ser. SINE1 contained the retroviral U5 region, whereas SINE2 did not. This is the first sequence-level demonstration of the existence of neighboring SINEs in medaka.     

Adaptation of the hypoosmotic swelling test to assess functional integrity of stallion spermatozoal plasma membranes

Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay consistency, accuracy, reliability and repeatability also were determined. Maximum spermatozoal plasma membrane swelling was observed in a 100 mosmol sucrose solution (P < 0.001). Incubation time (P = 0.67), temperature (P = 0.70) and total number of spermatozoa examined (P = 0.38 and P = 0.24 for 100 vs 200 and 100 vs 500, respectively) did not influence percent of HOS positive spermatozoa observed. A high degree of assay accuracy was indicated when a correlation of r = 0.998 was obtained between the HOS positive spermatozoa observed and expected when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. Assay consistency was demonstrated, as reflected by a mean coefficient of variation of 0.073 for 4 stallions. Also, the coefficient of variation from 2 analyses of variance was 0.168 and 0.096, indicating reasonably good assay reliability; estimates of repeatability from the same analyses were 0.794 and 0.968. The HOS test adapted to stallion spermatozoa in this study is a simple, highly accurate and consistent assay with good reliability and repeatability. Results observed under the conditions evaluated also permit some flexibility in adapting this assay to individual laboratory and practice settings for evaluating stallion spermatozoal plasma membranes.     

The controversy, potential and roles of spermatozoal RNA

The majority of cellular and molecular andrologists endorse the view that the sperm is a vessel for transporting the paternal genome to the waiting egg and nothing more. Any requirement for additional spermatozoal components that enter the ooplasm apart from the paternal centriole and the soluble egg-activating factor is generally dismissed. Many studies, however, have reported RNAs in ejaculate spermatozoa and we now know that mRNAs are delivered to the egg on fertilisation. The function and utility of sperm mRNA remains essentially unexplored. Here, we examine the controversy surrounding spermatozoal mRNA carriage, the evidence refuting its presence as an artefact and how spermatozoal mRNA is leading us to suspect that, quite apart from its undoubted diagnostic potential, it might have an important role in the establishment and maintenance of a viable paternal genome.     

Effects of cholera toxin and 5'-guanylylimidodiphosphate on human spermatozoal adenylate cyclase activity

The effects of cholera toxin and 5′-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoal adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 μg/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase assay, or was added to lysed spermatozoa, where the adenylate cyclase would be accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 μM was effective in activating human spermatozoal adenylate cyclase activity.