In. vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: Progesterone production by dispersed luteal cells

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term $\text{in vitro}$ studies of progesterone production by suspensions of collagenase-dispersed luteal cells in the presence and absence of exogenous gonadotropin (human chortonic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the incubation medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion above basal level ($\text{63.7 ± 13.1}\text{versus}\text{24.7 ± 5.5}\text{ng progesterone}\text{/}\text{ml}\text{/5 × 10}{}^4\text{cells}\text{/ 3}\text{hr}\text{,}\text{X}\text{± S.E.,}\text{n}\text{= 6;}\text{p}\text{< 0.05}$). However, luteal cells from early pregnancy (23–26 days after fertilization) secreted significantly less progesterone than cells of the non-fertile menstrual cycle (3.6 ± 2.4 versus 24.7 ± 5.5 ng/ml/5 × 10⁴ cells/3 hr, n = 3; p < 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108–118 days gestation) luteal cells exhibited partially renewed function, and near the time of parturition (163–166 days gestation) basal and HCG-stimulated progesterone secretion (30.2 ± 5.6 and 63.0 ± 13.0 ng/ml/5 × 10⁴ cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when steroidogenic activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitive steroidogenesis similar to that of the corpus luteum of the menstrual cycle.     

Effect of protected protein supplements on some testicular traits in Brahman cross bulls

Differences in a number of testicular traits were examined in 12 Brahman cross (F₂ generation $\text{1}\text{2}$ and $\text{3}\text{4}$ BX) bulls fed either poor-quality native pasture (NP) hay or NP hay with a protected protein supplement. Supplementation for 60 days significantly (P < 0.05) increased roughage dry matter intake (7.7 $\text{v}$ 5.6 kg/head/day), enabling maintenance of liveweight, whereas control animals lost 40 kg. There were significant (P < 0.05) decreases in scrotal circumference (1.5cm) and testicular consistency (0.8 score) in the control group, in which testes weights at slaughter were significantly (P < 0.05) less (373 g $\text{v}$ 459 g), with corresponding lower epididymal weights (37.4 g $\text{v}$ 43.5 g). Estimates of daily sperm production per gram (DSPG) were similar for both groups, and testis daily sperm production (DSP) was somewhat but not significantly (P>0.05) lower in the control group (4.3 × 10⁹$\text{v}$ 6.0 × 10⁹) as a result of lower testis weights. Total epididymal sperm storage capacity was also lower in control bulls (17.2 × 10⁹$\text{v}$ 27.0 × 10⁹), but only significantly (P < 0.05) in relation to cauda sperm reserves (8.5 × 10⁹$\text{v}$ 13.6 × 10⁹). Luteinizing hormone (LH) and testosterone responses to gonadotrophin releasing hormone (GnRH) treatment were similar for both groups, although LH responses to GnRH were greater in $\text{1}\text{2}$ BX than in $\text{3}\text{4}$ BX bulls.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?

The ability of the male gonad to convert androgens into estrogens is well known. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450_arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450_arom mRNA in adult rat using RT-PCR. With 2 highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but more importantly in highlyenriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450_arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cell (pachytene spermatocytes and round spermatids) preparation of the mature rat. After incubation with tritiated androstenedione, the aromatase activities in the microsomal fractions were 3.12±0.19 pmoles/mg/h in the testis, 1.25±0.13 in the seminiferous tubules and 1.53±0.15 in the crude germ cells. In purified testicular spermatozoa the aromatase activity was 2.96±0.69 pmoles/mg/h and found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450_arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from 90 day-old rats the P450_arom mRNA level was: 36.2±3.4×10^?3 amoles/μg RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450_arom mRNA levels were re pectively 367.2±76.6, 117.6±22.0 and <1×10^?3 amole/μg RNA. In conclusion we have demonstrated that the P450 aromatase is present not only in Sertoli cells and Leydig cells from mature rat testis but a biologically active aromatase exists also in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

The extrasplanchnic origin of blood dihydrotestosterone in elderly men

The splanchnic extraction and interconversion of testosterone (T) and dihydrotestosterone (DHT) were studied in 5 elderly men undergoing cardiac catheterization using a constant Infusion of [1,2-³H] testosterone and [4-¹⁴C] DHT. Metabolic clearance rate (MCR), splanchnic extraction (SE), splanchnic clearance (SC), extrasplanchnic clearance (ESC), transfer constant In blood ([P]_BB^T-DHT) and transfer constant across the liver ([P]_BB^T-DHT) were calc?ulated. The MCR^T was 675 ± 108 (mean ± SC) L/day and MCR^DHT was 409 ± 68 L/day. SE^T was 45.9 ± 7.0% and SE^DHT was 18.5 ± 5.4%. When these values are compared with those recently reported by us for normal men, there is a $\text{1}\text{3}$ reduction in SE^T and $\text{1}\text{2}$ reduction for SE^DHT in elderly men. The calculated SC^T and ESC^T were 355 ± 72 L/day and 320 ± 86 L/day, respectively. SC^DHT and ESC^DHT were 145 + 48 L/day and 263 ± 77 L/day respectively, suggesting that a major fraction of DHT is metabolized in extrasplanchnic organs. No evidence for a net appearance of DHT by either mass or specific activity analysis in hepatic vein blood was observed indicating that the splanchnic compartment does not contribute DHT into the circulation either by $\text{de novp}$ synthesis or via conversion from testosterone. This work indicates that conversion of testosterone to DHT in elderly men occurs entirely in extrasplanchnic tissue.     

Effects of lighting programs on onset of the ovulatory season in mares

Using 240 pony mares, lighting regimens were tested for their efficiency in hastening the onset of the ovulatory season. The mean number of days from January 1 to first ovulation was used as the end point. No advantage was gained by beginning a fixed lighting regimen ($\text{15.5L}\text{8.5D}$, hours light/hours dark) November 1 (66 ±8) versus December 1 (65 ±9), but beginning on January 1 was less efficient (98 ±8; controls, 132 ±5; P<0.05). In another experiment, daily three-hour interruptions of either the light phase (67 ±10) or the dark phase (71 ±11) did not significantly retard the effectiveness of a fixed regimen of $\text{15L}\text{9D}$ (54 ±5; controls, 142 ±6). A $\text{15L}\text{9D}$ regimen every other day (natural day length on alternate days) resulted in an interval (85 ±7) that was shorter (P<0.05) than for the controls and longer (not significant) than for the daily $\text{15L}\text{9D}$ regimen. When used with natural day length, a one-hour pulse of light in the evening (15 hours after sunrise) was not effective (141 ±6); a one-hour pulse in the morning 9.5 hours after sunset) was only partially effective (117 ±6). In another experiment, the interval was reduced (P<0.05) in a group with one hour of light fixed at 4:00 a.m. with natural day length (85 ±8; $\text{15L}\text{9D}$, 75 ±7; controls, 126 ±9). Results indicated that a fixed one-hour pulse of light at 4 a.m., used with natural day length, may provide an acceptable level of stimulation.     

The dimer-monomer equilibrium constant for [125I]β nerve growth factor

The equilibrium constant for $\text{[}{}^{125}\text{I]β}$ nerve growth factor was determined using polyacrylamide gel electrophoresis to separate the monomer and dimer. Various concentrations of the radiolabelled nerve growth factor were incubated for 24 and 48 hours. The equilibrium constants obtained for both incubation periods were the same, 3.2 ± 1.4 × 10^?11$\text{M}$ and 2.6 ± 1.6 × 10^?11$\text{M}$, respectively. Thus, at physiological concentrations the β nerve growth factor is in the dimeric form almost exculsively.     

Dopamine-stimulated cyclic AMP and binding of [3H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10^?8 M and was half-maximal at $\text{7.9±3.4·10}{}^{\mathrm{?7}}\text{M}$. The increase at 1·10^?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10^?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10^?5 M dopamine was $\text{2.3±0.9·10}{}^{\mathrm{?6}}\text{M}$. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value for high affinity binding sites was $\text{0.43±0.1·10}{}^{\mathrm{?7}}\text{M}$ and $\text{4.7±1.6·10}{}^{\mathrm{?7}}\text{M}$ for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was $\text{1.2±0.4·10}{}^{\mathrm{/t-7}}\text{M}$ noradrenaline, 2·10^/t-7 M epinine, $\text{4.1±1.8·10}{}^{\mathrm{/t-6}}\text{M}$ fluphenazine, $\text{8.0±1.6·10}{}^{\mathrm{/t-6}}\text{M}$ haloperidol, $\text{4.2±1.2·10}{}^{\mathrm{?6}}\text{M}$cis-flupenthixol, $\text{2.7±0.4·10}{}^{\mathrm{?5}}\text{M}$trans-flupenthixol, $\text{>1·10}{}^{\mathrm{?5}}\text{M}$ apomorphine, sulpiride, naloxone and isoproterenol.     

Changes in basic chromosomal proteins during spermatogenesis in the mature rat.

Nuclei of the seminiferous epithelial cells of rat testis were filtered through glass wool to remove sperm heads, flagellae and late-stage spermatids and then centrifuged through sucrose gradients to yield three fractions. The cellular origins of the predominant nuclei in these fractions were identified through the kinetics of labeling with [³H]thymidine. The relative amounts of the different histone fractions changed during the various stages of spermatogenesis in an interesting and systematic manner. For example, the ratio of the trailing (acetylated) to the leading member of the histone F2a1 doublet was greater in spermatid nuclei than in nuclei of a fraction enriched in primary spermatocytes. Similarly, the ratio $\text{X}{}_1\text{F}\text{1}$ was greatest in spermatid nuclei. On the other hand, the ratio $\text{X}{}_3\text{F}\text{2b}$ was greater in the nuclei of pachytene-diplotene primary spermatocytes than in the fraction enriched in nuclei of spermatogonia and preloptotene primary spermatocytes.A basic protein fraction with some of the properties of a protamine was extracted from rat sperm heads and from the nuclei of spermatids. This protein fraction has high contents of arginine and cysteine (after reduction), and it appears to be identical with the protamine described by Kistler et al. In addition, a new protamine was isolated from rat sperm heads which has high arginine content but appears to be devoid of lysine and cyst(e)ine. Two other basic protein fractions with high electrophoretic mobilities were extracted with acid from the nuclei of testicular seminiferous epithetial cells without prior reduction. One of these proteins may be identical with the testis-specific protein of Kistler et al.     

Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

The DNA containing the genes for rRNA (commonly called rDNA) of $\text{Tetrahymena}$ sediments in sucrose density gradients considerably slower than the main part of the DNA when DNA from gently lysed whole cells or isolated nuclei are fractionated by this method. In rDNA purified by CsCl gradient centrifugation about 20% of the DNA (40% of the bases in one strand) consists of sequences homologous to 25S and 17S rRNA as determined by DNA-RNA hybridization. The purified rDNA co-sediments in sucrose gradients with Ø29 phage DNA (M.W. = 11 × 10⁶). Examination by electron microscopy of the rDNA demonstrates that the molecules are linear with a length of 5.65 ±0.6 μm corresponding to a molecular weight of 11 × 10⁶.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

The aging leydig cell: VII. Cytoplasmic estradiol receptors

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2–3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 ± 0.3 fmol/mg protein ($\text{x}\text{±}\text{SE, n}\text{=12}$), which was comparable to that of the old rats, 5.7 ± 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 × 10¹⁰M^?1. Plasma estradiol levels were also similar in both groups-19.6 ± 2.8 pg/ ml (n=14) for the young and 19.2 ± 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.     

Halogenated biphenyls as AHH inducers: Effects of different halogen substituents

4′-Iodo-, 4′-bromo-, 4′-chloro- and 4′-fluoro-2,3,4,5-tetrachlorobiphenyl were administered to immature male Wistar rats and the effects of this homologous series of 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal drug-metabolizing enzymes were determined. All the halogenated biphenyls increased microsomal benzo[a]pyrene hydroxylase (or aryl hydrocarbon hydroxylase, AHH), ethoxyresorufin (ER) O-deethylase and dimethylaminoantipyrine (DMAP) N-demethylase. The effects of the 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal enzyme activities and on the relative peak intensities and spectral shifts of the reduced cytochrome P-450:CO and ethylisocyanide (EIC) binding difference spectra were similar to those observed after coadministration of phenobarbitone (PB) and 3-methylcholanthrene (MC). The relative activities of the halogenated biphenyls were determined using two $\text{in}$$\text{vitro}$ assays; namely cytochrome P-448 associated induction in rat hepatoma H-4-II E cells in culture and competitive binding to the hepatic cytosolic $\text{Ah}$ receptor protein from male Wistar rats. Dose-response experiments for the iodo, bromo, chloro and fluoro analogs gave EC₅₀(M) values of 8.5×10^?9, 6.6×10^?8, 5.7×10^?7, and 3.3×10^?5, and 1.5×10^?6, 2.5×10^?6, 4.1×10^?6 and 2.5×10^?5 for the ER O-deethylase induction and receptor binding assays respectively. The relative potencies of the 4′-halo-2,3,4,5-tetrachlorobiphenyls followed the order I>Br>Cl>F for both assays and differences in the EC₅₀ values for the iodo and fluoro analogs were greater than three orders of magnitude for ER O-deethylase induction in rat hepatoma cells in culture. One possible explanation for these effects may be associated with differences in the polarizability of the laterally substituted halogen groups. However, other differences in the physico-chemical properties of the halogen atoms may also be important.     

Fertility of ram semen after storage at 5°C

In five experiments, fertilization, early (18–19-day) pregnancy, and lambing were examined after insemination with semen stored at 5°C in tris-fructose-egg yolk diluent.After deposition into uterine horns by surgical insemination of semen stored for 0 (control), 2, 4, 6, 8, 9 or 10 days, fertilized eggs were recovered in $\text{32}\text{34}\text{,}\text{16}\text{16}\text{,}\text{21}\text{22}\text{,}\text{15}\text{20}$, $\text{9}\text{17}\text{,}\text{2}\text{18}$ and $\text{1}\text{15}$ ewes; the 18–19-day pregnancy rates determined by progesterone assay were $\text{32}\text{48}\text{,}\text{15}\text{28}\text{,}\text{11}\text{20}\text{,}\text{12}\text{20}\text{,}\text{9}\text{20}$, $\text{2}\text{20}$ and $\text{1}\text{21}$ for the respective storage periods. There was a linear decrease in fertilization rates beyond 4 days of storage and in early pregnancy rates after 6 days of storage (P<0.001). The decline with time of storage in the fertilization rate was not associated with an increase in early embryonic loss. Surgical insemination with semen stored for 0, 4, 6, 8, 9 and 10 days resulted in 53, 35, 40, 25, 5, and 0% lambing.Single cervical (normal) insemination of a total of 281 ewes with 0, 1, 2 or 3-day-old semen, using within each semen treatment 90 × 10⁶ and 180 × 10⁶ spermatozoa, yielded mean lambing rates of 60.0, 34.3, 33.8, and 17.1%; and after using 150 × 10⁶ and 300 × 10⁶ spermatozoa in a total of 393 ewes the mean lambing rates for the above semen treatments were 69.0, 46.4, 36.1, and 24.2% (linear, P < 0.001). In both tests the lambing results were better after insemination of the higher number of spermatozoa, but the slope of decline in fertility with age of semen was not affected by the sperm dose.When single and double cervical inseminations were performed in a total of 411 ewes, with 150 × 10⁶ and 300 × 10⁶ spermatozoa per inseminate, the lambing rates for semen stored for 0, 1, 2 and 3 days were 57.7, 30.4, 26.8, and 4.7% after single insemination, and 66.7, 56.8, 46.4, and 41.5% after double inseminations. The sperm dose within method of insemination and semen treatment had no effect. The lambing rate was better after double than single insemination (P<0.001), but the slope of decline in fertility with age of semen was not significantly affected by number of inseminations.In the final experiment, involving 408 ewes, 300 μg of prostaglandin F₂α added to the inseminate did not improve the fertility of fresh semen or semen stored for 1 day.     

Molecular characterization of a stable Flac plasmid

F$\text{lac}$S is a thermostable extrachromosomal element isolated in $\text{Salmonella typhimurium}$ which is altered in its replication as compared to its precursor F_ts114$\text{lac}$. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F_ts114$\text{lac}$ to be 81 × 10⁶ daltons while that of F$\text{lac}$S is 109 × 10⁶ daltons. F$\text{lac}$S may carry a segment of $\text{S. typhimurium}$ chromosomal or cryptic plasmid DNA.