Spermatogenesis does not require the local production of follistatin.

It has been proposed that follistatin can modulate the actions of activins and/or other members of the transforming growth factor-beta superfamily of proteins on testicular function, since mice overexpressing follistatin showed spermatogenic disruption. However, since mice with targeted disruption of the follistatin gene die soon after birth, it is not feasible to determine the effect of the absence of follistatin on testicular function using this model. To further understand the role of follistatin on the development and maintenance of spermatogenesis, fetal testes, collected by Caesarean section at day 18 of gestation from follistatin null mice, were transplanted to the external ear of castrated recombination activating gene 1 immunocompromised male mice. The testicular grafts were then analysed 7-8 weeks after transplantation and showed that full spermatogenesis developed in both the testes of wild-type and follistatin null mice. This study indicates that, if follistatin is required to modulate spermatogenic development, it is not supplied by local testicular production but by circulating follistatin from the host mouse.

The role of activin, follistatin and inhibin in testicular physiology.

The role of the inhibins, activins and follistatins in testicular function are being more clearly defined following studies describing the cellular localisation of these proteins to the testis and the availability of specific assay systems enabling measurement of these proteins. Taken together with the results of targetted gene inactivation experiments, several concepts emerge. Inhibin B is predominantly produced by the Sertoli cell in many adult male mammals whereas there is a perinatal peak of inhibin A in the rat. In contrast, activin A has its highest concentrations in the immediate post-natal period during which it is involved in the developmental regulation of both germ cells and Sertoli cells being modulated by follistatin. Activin A levels are considerably lower in the adult testis but Sertoli cell production is stimulated by interleukin-1 and inhibited by FSH. Little is known about the production of activin B due to the absence of a suitable assay but the beta(B) subunit mRNA is expressed in germ cells and Sertoli cells and is stage-dependent. This pattern of expression suggest that it may be involved in autocrine or paracrine actions within the seminiferous epithelium.

Parthenogenetic activation of rat oocytes and their development (in vitro).

The present study was performed to determine suitable methods for parthenogenetic activation and subsequent development of rat oocytes in vitro. In the first series of experiments, the ability of electrical pulses, strontium, ethanol and ionomycin to activate Sprague-Dawley (SD) rat oocytes was examined. The synergistic effect of strontium and cycloheximide or puromycin was also examined in the second series of experiments. In the third series of experiments, the development of F1 hybrid (SD x Dark Agouti) parthenotes activated with different concentrations of strontium (10-0.08 mM) was compared with that of SD parthenotes. The effect of the timing of activation (10 min and 2, 4 and 6 h after cervical dislocation) was also assessed in a fourth series of experiments. The oocytes activated by strontium showed higher pronuclear formation and cleavage rates than those in the other groups (P < 0.05). Higher blastocyst development was obtained from parthenotes activated by strontium and strontium-cycloheximide compared with the strontium-puromycin group (P < 0.01). However, the total cell number of blastocysts from the strontium-cycloheximide activation group was higher than that of other groups (P < 0.05). With strontium (2.5-10 mM) treatment, 40.9% of blastocysts were obtained from F1 hybrid oocytes, whereas 22.9% were obtained from SD (P < 0.01). The oocytes activated 10 min or 2 h following cervical dislocation showed higher blastocyst development than those of the 4 and 6 h groups (P < 0.01). These results suggest that strontium-cycloheximide produces the highest parthenogenetic activation rate in the rat and that oocytes must be activated by 2 h after cervical dislocation.

Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH.

Previous studies indicate that proliferation of rat Sertoli cells in culture can only be maintained until the equivalent of days 10-12 after birth, irrespective of the age of the donor animal. This report describes methods for the isolation and culture of Sertoli cells from day 6 rat testes, which can proliferate in culture for 20-24 days (that is, until the equivalent of days 26-30 after birth). Cells were isolated by enzymatic digestion of seminiferous cords followed by selective depletion of contaminating peritubular cells by adhesion to a polystyrene surface. The purity of the Sertoli cells was assessed using a combination of markers to be > 99.5%. Proliferation was assayed using tritiated thymidine incorporation and further verified by bromodeoxyuridine histochemistry and flow cytometry. Sertoli cells proliferated at basal levels in Dulbecco's modified Eagle's medium (DMEM)-F12 media alone, and proliferation was stimulated further by addition of recombinant human FSH to the culture media. After 20-24 days in culture, proliferation rapidly ceased, and cells assumed abnormal morphology and detached from the culture vessel; these events are consistent with the cells undergoing classic rodent cell senescence. The method described provides a useful tool for investigating the control of Sertoli cell division. Furthermore, these findings indicate that the timely differentiation of Sertoli cells is not dependent solely on an intrinsic timing mechanism, as has been suggested previously.

Developmental expression of thyroid hormone receptors in the rat testis.

Sertoli cell proliferation in the rat is completed by Days 15-20 postnatally. Thyroid hormones appear to regulate the duration of Sertoli cell proliferation, affecting adult Sertoli cell number and hence the capacity of the testis to produce sperm. In the present study, a combination of immunohistochemistry, immunoblot analysis, and reverse transcription-polymerase chain reaction was used to demonstrate the expression pattern of thyroid hormone receptors (TR) in the juvenile and adult rat testis. The results indicated that TRalpha1 was expressed in proliferating Sertoli cell nuclei, its expression decreasing coincident with the cessation of proliferation. TRalpha2, TRalpha3, and TRbeta1 mRNAs were expressed at low levels during development; however, the corresponding protein was not detected by immunoblot analysis. In addition, TRalpha1 was found to be expressed in germ cells from intermediate spermatogonia to mid-cycle pachytene spermatocytes. Immunohistochemistry also demonstrated TR expression in a subset of interstitial cells. The demonstration of TR expression in germ cells undergoing spermatogenic differentiation suggests a possible role for thyroid hormones in the adult testis.

Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.

Cholesteryl ester transfer between high density lipoprotein and phospholipid bilayers.

High density lipoprotein-associated cholesteryl esters (HDL CE) are taken up by many cells without parallel uptake of HDL apoproteins, a pathway we have termed "selective uptake." The first step in this pathway, the reversible incorporation of HDL CE into the plasma membrane, is the subject of the present study. To examine the role of membrane proteins, the rate of HDL CE incorporation into isolated rat liver plasma membrane was compared with the rate of incorporation into synthetic membranes devoid of protein. Both membrane systems exhibited saturable uptake of CE, and at rates that were similar (t1/2 approximately 2 h, measured with 50 micrograms of HDL protein). Addition of unlabeled HDL to "chase" CE tracer from the biological and synthetic membranes revealed two kinetically distinct CE pools (t1/2 approximately 0.5 h and t1/2 approximately 30 h). Both biological and synthetic membranes accepted similar amounts of CE into both pools, with a maximum incorporation of 2-4 mol % relative to membrane phospholipids. CE transfer between HDL and membranes was kinetically second-order, in contrast to the first-order transfer of unesterified cholesterol. There was no evidence for direct participation of any apolipoprotein in CE uptake; CE in HDL or in protein-free microemulsions of similar particle size transferred to membranes at similar rates. To examine the possibility that CE transfer requires transient fusion of the HDL amphipathic coat with the membrane outer leaflet, radiolabeled cardiolipin was incorporated into either HDL particles or into synthetic membranes as an amphipathic coat marker that does not diffuse through the aqueous phase; transfer between HDL and membranes was not observed. Thus, CE are transferred between HDL and cell membranes in a collision-mediated process that does not involve amphipathic coat fusion and is not dependent on either membrane protein or apolipoproteins.

The apolipoprotein B messenger RNA editing enzyme.

The editing of apolipoprotein (apo)B messenger RNA (mRNA) involves a novel C to U modification, which creates an in-frame stop-translation codon, thereby generating the carboxyl-terminal of apoB48. The 27 kDa catalytic subunit of the editing enzyme has been cloned and established to be a zinc-containing cytidine deaminase. The catalytic subunit is guided to the editing site by a second targeting subunit or subunits. A candidate for the targeting subunit is a 60 kDa protein that can be UV crosslinked to the sequence UGAU, which is part of a motif downstream of the editing site that is essential for editing.

The p27 catalytic subunit of the apolipoprotein B mRNA editing enzyme is a cytidine deaminase.

The messenger RNA for apolipoprotein B undergoes a discrete and specific C to U editing of nucleotide 6666. This generates a stop translation codon and defines the carboxyl terminus of apolipoprotein B48. A 27-kDa rat intestinal protein that does not itself edit apolipoprotein B mRNA, but confers editing activity on chick intestinal extracts that do not have intrinsic editing activity, has recently been identified and its cDNA cloned (Teng, B., Burant, C. F., and Davidson, N. O. (1993) Science 260, 1816-1819). Here we show that p27 is homologous in the zinc coordinating region of the active site to cytidine deaminases from Escherichia coli, Bacillus subtilis, yeast, and man and to deoxycytidylate deaminases from T2 and T4 bacteriophages and man. p27 expressed in Xenopus laevis oocyte extracts has cytidine deaminase activity and specifically confers editing activity on chick intestinal extracts. The homologous E. coli cytidine deaminase does not confer editing activity. The zinc-specific chelating agent o-phenanthroline abolishes p27 activity and site-specific apolipoprotein B mRNA editing in rat enterocyte editing extracts. We conclude that p27 is the catalytic subunit of the apolipoprotein B mRNA editing enzyme and is a zinc-containing cytidine deaminase.

Monoclonal antibodies to human apolipoprotein AI: probing the putative receptor binding domain of apolipoprotein AI.

We have used four monoclonal antibodies (MAbs) specific for human apolipoprotein (apo) AI, designated AI-1, AI-3, AI-4.1 and AI-4.2, to study the interaction between high-density lipoprotein HDL3 and rat liver plasma membranes. MAbs AI-1 and AI-3 recognize epitopes within residues 28-47 and 140-147 respectively of apoA-I [Allan, Tetaz and Fidge (1991) J. Lipid Res. 32, 595-601]. Two previously unreported MAbs, AI-4.1 and AI-4.2, were raised against purified CNBr fragment 4 (CF4) of apoAI, the C-terminal region. Using e.l.i.s.a. and immunoblotting techniques, we have demonstrated that all four MAbs recognize distinct epitopes within apoAI. Epitope mapping studies using endoproteinase cleavage peptides of CF4 showed that AI-4.1 binds to an epitope within residues 223-233, which is poorly exposed on apoAI molecules associated with lipid. Fab fragments derived from MAb AI-4.2 inhibited the binding of 125I-labelled HDL3 to rat liver plasma membranes, whereas Fab fragments from AI-4.1, AI-3 and AI-1 had little or no effect. In ligand blotting studies with purified CNBr fragments of apoAI and using apoAI-specific antibodies for detection, CF4 showed the highest capacity to recognize two HDL-binding proteins previously identified in rat liver plasma membranes. We propose that the specific interaction between HDL and liver plasma membranes is largely mediated through a binding domain in the C-terminus of apoAI, which is consistent with the involvement of specific receptors for the apolipoprotein moiety of HDL.

Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein.

There is little dispute that high density lipoprotein (HDL) binds to cells, however, the nature of the interaction is not fully understood. We now present evidence for a new binding site of higher affinity but lower capacity than the sites previously described in the literature. This new site is characterized by high affinity/low capacity for HDL binding (Kd = 0.94 microgram/ml, Bmax = 36 ng/mg), while the low affinity site (Kd = 36 micrograms/ml, Bmax approximately 700 ng/mg) appears to be consistent with the literature values for the interaction of HDL with cells and isolated membranes. Proteolysis of HDL with trypsin abolished its interaction with the high affinity site, suggesting an apolipoprotein requirement, while having no effect on binding to the lower affinity site. Kinetic rates of association/dissociation were determined in order to further characterize the high affinity site. At a concentration which favored the binding of HDL with the high affinity site (1 microgram/ml, 37 degrees C), the time course of association of HDL with rat liver plasma membranes, displayed a biphasic pattern, requiring 6-8 h to reach the level of binding predicted from the saturation studies. The second phase was highly sensitive to temperature, being considerably slower at 24 degrees C and totally abolished at 0 degrees C. A kinetic Kd, derived from the measured association and dissociation rate constants (Kd = 0.31 microgram/ml), was found to be of a similar magnitude to the Kd calculated for the high affinity site by Scatchard analysis (Kd = 0.94 microgram/ml). In summary, the high affinity site on rat liver plasma membranes displays an apoprotein requirement and kinetic parameters, consistent with a ligand-receptor interaction.

Relaxed specificity of endoproteinase Asp-N: this enzyme cleaves at peptide bonds N-terminal to glutamate as well as aspartate and cysteic acid residues.

Asp-N, an endoproteinase specific for cleavage of protein or polypeptide bonds N-terminal to aspartate or cysteic acid residues, has been shown to possess a similar affinity for certain glutamate residues. Of 18 glutamate residues present in 2 cyanogen bromide fragments of apolipoprotein A-I, 5 residues were cleaved at rates comparable to that of cleavage at the 12 internal aspartate residues present in these polypeptides (all of which were cleaved). Cleavage of these 5 glutamate residues was obtained under standard enzyme digestion conditions, and the identities of all peptides obtained by Asp-N digestion were determined by amino acid sequencing of peaks obtained from reversed-phase high performance liquid chromatography.

Studies on the formation, separation, and characterization of cyanogen bromide fragments of human AI apolipoprotein.

We have sought to obtain conditions for cyanogen bromide (CNBr) cleavage of apolipoprotein AI which would preserve, as far as possible, the biological activity of the resulting fragments. We found that the choice of solvent is an important consideration since modification of amino acids in different proteins varies with cleavage conditions. Initially, an analytical technique employing reversed-phase (RP)-HPLC which separates the four CNBr fragments in a single chromatographic step was established to monitor the products and extent of cleavage. In developing this technique, spectral data indicated damage to tyrosine and tryptophan residues during CNBr digestion. This problem was resolved by using 70% trifluoroacetic acid instead of 70% formic acid as the solvent, which had the added benefit of increasing the extent of cleavage of the Met86-Ser87 bond by 50%. We applied the information derived from the analytical RP-HPLC method to achieve the preparative isolation of CNBr fragments. This procedure included a gel permeation chromatography step using a citrate/urea buffer before RP-HPLC to isolate pure fragments in volatile buffers. Finally, we discuss aspects of structural integrity with an emphasis on modification of aromatic amino acids and deamidation of asparagine and glutamine residues.

Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy.

Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter time constant was attributed to motion of the chromophore within the lipoprotein particle, while the longer time constant represented the global tumbling of the particles in solution. The measured correlation times for this global motion were about twice those predicted from the Stokes-Einstein relationship. Covalent labeling of the apolipoproteins of the low and high density lipoproteins with erythrosin revealed the existence of segmental motion of labeled domains of the apolipoprotein within their respective particles. The correlation times for this motion were within the range 10-50 microseconds. The binding of low density lipoproteins to receptors on membranes isolated from the adrenal cortex resulted in a freezing of the global motion, but maintenance of the faster segmental motion of the labeled domains of the apolipoprotein. The experiments imply that in these membranes there is no global motion of the low density lipoprotein-receptor complex on the phosphorescence time scale. Similar results were found for the binding of high density lipoproteins to liver plasma membranes. The contributions of nonspecific binding of the labeled lipoproteins to the measured phosphorescence anisotropy were carefully assessed.

Identification of Actinomyces (Corynebacterium) pyogenes with the API 20 Strep system.

A total of 62 strains of Actinomyces pyogenes (previously Corynebacterium pyogenes) were examined by the API 20 Strep system (API System, La Balme Les Grottes, Montalieu-Vercieu, France). The system was shown to be reliable and rapid when the tests were compared with standard identification methods. No confusion occurred with streptococcal profiles in the current API 20 Strep data base.

Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity.

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.

Causes and treatment of insomnia among adolescents.

Though the preponderance of research on insomnia suggests that this malady occurs primarily among the adult population, recent research has revealed that as many as 13% of the adolescent population may suffer from chronic insomnia. Having one in 10 of the school population afflicted by this seldom considered ailment holds serious implications for learning. Adolescent insomnia can impair the victim's daily existence and affect personal life, school performance, and school attendance. Furthermore, if not dealt with initially, adolescent insomnia may lead to adult insomnia and dependence on alcohol and other drugs. The prevalence of adolescent insomnia, its cause, diagnosis, and treatment are examined.

Early birth order in Briquet's syndrome.

Birth order in 90 female patients with Briquet's syndrome was significantly earlier than the theoretical mean for a normally distributed population. In contrast, the birth order of 78 women with primary alcoholism did not differ from this mean. These results suggest an early environmental influence in the development of Briquet's syndrome in women.