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BACKGROUND: Low-level Laser Therapy (LLLT) has demonstrated its potential in promoting fiber matrix maturation, collagen synthesis, and fibroblast proliferation, contributing to tissue regeneration. Our study aimed to investigate the impact of LLLT on collagen type I synthesis, cell proliferation, and viability in human ligament fibroblasts derived from the Anterior Cruciate Ligament (ACL). METHODS: Tissue samples were obtained from individuals undergoing arthroscopic ACL reconstruction surgery. Primary human fibroblasts were isolated, and immunohistochemical assays confirmed their characteristics. LLLT at 850 nm was administered in three groups: Low dose (1.0 J/cm²), High dose (5.0 J/cm²), and Control (0.0 J/cm²). Cell viability was calculated using a membrane integrity assay, proliferation was determined by automated counting, and collagen type I concentration in cell culture was measured using an immunoassay. RESULTS: Fibroblasts showed decreased viability after low and high doses of LLLT, increased proliferation at the low dose, and increased collagen synthesis at the high dose on day 10 for both sexes after treatment. CONCLUSION: Our study demonstrated that LLLT may improve the early ligament healing process by increasing cell proliferation at the low dose and enhancing collagen type I synthesis at the high dose in human ligament fibroblasts.

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The understanding of the tribological behavior of natural structures has been used as inspiration to design and optimize surfaces for diverse applications in engineering. In the present work, morphological, microstructural, mechanical and tribological characterization of the shed skin of two snake species, namely Boa Red Tail and Python Regius was carried out. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) analyses showed the existence of deterministic patterns, i.e., ordered arrays of geometrical features at the surface, while Transmission Electron Microscopy (TEM) allowed studying the internal structure and chemical composition of the skin sheds. Nanoindentation measurements showed significant variations in hardness and elastic modulus from the surface to the inner layers of the skin, and pin-on-disc tests revealed anisotropic behavior of the friction coefficient (COF) as a function of the sliding direction against balsa wood in dry conditions. Correlations between the friction data, nano-indentation mechanical properties and subsurface skin structure were established for both species taking into account the ways in which the skins' deterministic patterns influence the tribological performance.

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The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.

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Metalloproteinases are an important group of hydrolytic enzymes which participate in interstitial matrix degradation during tissue remodelling processes and therefore may be required during follicular growth and maturation. The activity of metalloproteinases (collagenases, gelatinase, and Pz-peptidase), was measured during growth, maturation and atresia of goat antral follicles. These follicles (n = 67) were separated by size and also classified into four groups: non-atretic (Group I); early atretic (Stage I) (Group II); moderately atretic (Stage II) (Group IIIa); and, late atretic (Stage III) (Group IIIb). Pz-peptidase was greater in granulosa than in thecal cells, and almost absent in follicular fluid. In non-atretic follicles, activity in granulosa cells increased with increasing follicle size, whereas activity peaked in 3-6 mm follicles in thecal cells. Atresia was associated with declining activity in thecal cells from follicles in the 3-6 mm range and in granulosa cells from the > 6 mm range. Interstitial collagenase activity was significant and similar in granulosa and thecal cell extracts and low in follicular fluid from non-atretic follicles. Activity increased significantly in thecal cells, but decreased significantly in granulosa cells from large (> 6 mm) non-atretic follicles. Atresia was associated with declining activity in both types cells and increasing activity in follicular fluid. Gelatinase activity was some times associated with five regions corresponding to molecular weights of 22.1, 30.7, 39.6, 63.8 and 71.4 kDa, and rarely at 91.3 and 81.2 kDa. Overall activity declined with atresia in thecal cells from follicles in the 3-6 mm range, but not in those > 6 mm. In granulosa cells from follicles 3-6 mm, activity varied widely with stage of atresia, while in cells from follicles > 6 mm, activity was greatly increased in atretic follicles.

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Galanin is a 29 amino-acid peptide originally isolated from porcine intestine. It is synthesized as part of a large precursor peptide the preprogalanin. Immunological studies has showed that there is interspecies conservation of the N terminal portion although the C-terminal portions has a little of immunoreactivity. Galanin has a number of pharmacological properties in whole animals and isolated tissues. Galanin contracts isolated preparation from rat fundus, ileum, colon and urinary bladder. Direct administration of galanin (pGal) into the rat third ventricule stimulates food intake, increases plasma growth hormone and prolactin levels and decrease dopamine levels in the median eminence. Intravenous infusion in dog and humans induce a hyperglycemia and glucose intolerance and inhibits the insulin, somatostatin and pancreatic polipeptide secretion from pancreas. Galanin is a estrogen-stimulated peptide. Estrogens increase dramatically the synthesis of their mRNA and the peptide in the rat pituitary. Galanin-like immunoreactivity is widely distributed in several mamalian species including humans. In the central nervous system it was found in medium emminence, hypothalamus, arcuate nucleus etc. Its localization in neurosecretory granules suggest that galanin functions as a neurotransmitter. The detection of a Gal-immunorectivity in the plasma after 17 beta estradiol stimulation suggests that galanin has a distal target and therefore, may be an additional anterior pituitary hormone. Galanin has been localized in reproductive tissues and this suggests that it may play an estrogen mediated role in the hypothalamic and pituitary function. However, the molecular mechanisms involved in their function remain to be studied.

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Large superovulatory doses of gonadotrophins result in reduced fertility in laboratory and large domestic animals and it has been postulated that some of the superovulated oocytes are derived from abnormal follicles which would not ovulate under normal physiological stimuli. Follicular growth, follicular maturation and atresia, ovulation and the nidation of the fertilized oocyte require intense tissue remodelation which can be accomplished only through the action of hydrolytic enzymes. We have studied the activities and sub-cellular distribution of three lysosomal enzymes (acid phosphatase, N-acetyl-beta-D-glucosaminidase and beta-glucuronidase) in the follicular fluid, granulosa and theca cells of preovulatory follicles and in the endometrial tissue of immature Wistar rats injected with 4 (control) or 40 (superovulated) IU of pregnant mare serum gonadotrophin (PMSG). Enzyme activities were from four to ten times higher in theca than in granulosa cells. This difference was particularly important in the case of beta-glucuronidase. Large preovulatory follicles tended to have higher activities of lysosomal enzymes in the free fraction of all the compartments studied. This difference was remarkable in theca cells where free enzymes would be required to help ovulation. Forty IU of PMSG induced higher activities of acid phosphatase in theca and granulosa cells than 4 IU, but in endometrial tissue this latter dose of PMSG was more efficient to induce higher activities of this enzyme. The endometrial bound fraction of N-acetyl-beta-D-glucosaminidase was almost three times higher than the free activity. This behavior was also observed in endometrial beta-glucuronidase but only in the control rats. The results observed in follicular fluid were less homogeneous. The activities of glucosaminidase and acid phosphatase were two to three times higher in rats overstimulated with 40 IU of PMSG than in the control rats, whereas the activities of beta-glucuronidase were lower in the superovulated rats. Our results suggest that alterations in the process of tissue remodeling required for ovulation of mature, normal oocytes and for nidation of the fertilized ovum may be important factors to explain pregnancy failure in the PMSG superovulated female.

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Modifications in the basal molecular biology parameters (total concentrations of RNA, DNA, proteins, rRNA, tRNa, free and polysomal bound poly-A+ mRNA) have been determined daily in the growing hypothalamus of male and female rats from day 1 to day 8 after birth. Changes observed in the parameters studied in this work occurred mainly in the first 48 h after birth. In males tRNA and free mRNA (f-mRNA) contents decreased from day 1 to day 2 and then their concentrations remained more or less constant. Total mRNA significantly decreased from day 1 to day 3 and showed a further significant decrease from day 6 to day 8. Polysomal bound-mRNA (b-mRNA) decreased from day 1 to day 3, then increased to day 6, and finally decreased once more from day 6 to day 8. The b-mRNA/f-mRNA, mRNA/rRNA and mRNA/total RNA ratios showed a bimodal behavior with a first peak on day 2, and a second, smaller peak, on days 6-7. The changes observed in the females on the first 2-3 days of life were the inverse of those observed in the males, most of the parameters studied showed a sharp increase from day 1 to day 2 or to day 3 and then a drastic decrease. The only exception to this behavior was the b-mRNA/f-mRNA ratio which showed a small decrease from day 1 to day 2, followed by a continuous increase from day 2 to day 8. b-mRNA concentrations, after the sharp decrease from day 2 to day 3 of life, increased from day 3 to day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

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Binding of N-formyl-methionyl-L-leucyl-[3H]phenylalanine (fML[3H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (Kd = 12.3 +/- 0.5 nM, n = 22,285 +/- 65,008 binding sites per sperm cell) and a second one (Kd = 700 +/- 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (Kd = 6.4 +/- 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.

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The structure of human sperm chromatin compared with somatic chromatin (liver) was studied by titration of the exposed DNA-phosphate groups with poly-1-lysine (3000 and 28,100 MW) and by their susceptibility to the hydrolytic action of micrococcal nuclease and DNase I. With both sizes of polylysine used, the binding values were significantly lower for sperm chromatin (0.31 +/- 0.05) than for liver chromatin (0.52 +/- 0.05), indicating the presence of about 30% and 52% of free phosphate groups, respectively. Interaction with liver chromatin left no polylysine molecules partially unbound ("wastage") even when 28,100 MW polylysine was used; on the contrary, sperm chromatin showed 26% of "wasted" polylysine even when the smaller polymer was used, indicating that in sperm chromatin the accessible DNA zones are usually no longer than 42 A, that is, 12 base pair. Sperm chromatin was notably more susceptible to both micrococcal nuclease and DNase I action than liver chromatin. However, in the presence of saturating concentrations of polylysine they were similarly protected. Micrococcal nuclease and DNase I hydrolysis products of sperm fractions when submitted to electrophoresis produced a polydisperse smearing pattern along the gel that was difficult to correlate with the presence of nucleosomal structure.

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Using the model of exchange transport, we found that glucose transport through the human spermatozoa membrane (447 +/- 54 pmoles/min/10(8) cells) is probably the rate-limiting step in sugar utilization. Sugar transport was more efficient for glucose than for fructose (182 +/- 32 pmoles/min/10(8) cells) and depends on a highly asymmetric carrier with at least two transporting sites. Transport was drastically dependent on pH with an optimal pH of 7.4, showing a decrease of more than 60% with a change of 1 pH unit. Testosterone and 17-B estradiol increased the amount of transported sugar (619 +/- 73 and 922 +/- 110 pmoles/min/10(8) cells, respectively), while progesterone has no effect.

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PIP: This study investigates the mechanism of action of the progesterone-releasing IUD. 10 volunteer women aged 25 to 35 years (UPS-65) were selected to use the IUD progesterone-releasing system for more than 4 years (54.4 +or- 2 months); all had regular menstrual cycles of 28 +or- 2 days. 15 untreated normal volunteers with a similar IUD system comprised the control. Endometrial samples were obtained from treated and control groups during the luteal phase (day 20 +or- 1) according to a previously published method. The samples were homogenized and divided into 2 aliquots. The first aliquot was used to measure DNA, RNA and proteins by methods previously described while the second was used for the determination of protein and RNA synthesis. The endometria of the UPS-65 group showed significant reductions in both total and per cell contents of proteins and RNA, compared with those of the controls. The DNA content however appeared not to be modified even after prolonged use of this contraceptive system. Analysis of nuclear (DNA) and cytoplasmic (RNA) mass and of cell size (prot/DNA) showed that while nuclear mass remained constant in the IUD users, functional mass (cytoplasmic mass (RNA) + cell size (prot/DNA)) was drastically reduced in this group of women. The RNA/DNA ratio (cytoplasmic mass per nuclear mass), a crude index of cytoplasmic metabolic capacity, was reduced by more than 50% in the endometrium of users. However, the actual functional activity per cell (RNA/protein) is preserved in the treated endometrium. A comparison of values for the incorporation capacity of radioactive leucine and uridine obtained from normal endometrium and from treated endometrium (UPS-65 group) showed significant lowered values in the treated endometrium from 16.7 +or- 5.7 to 4.1 +or- 2.0 pmol leucine/mg protein for protein synthesis and from 5.1 +or- 2.0 to 2.2 +or- 1.5 pmol uridine/mg protein for RNA synthesis. The mode of action of the progesterone releasing IUD involves a change in both the biochemical composition and in the macromolecular metabolism of the endometrium. Modifications of sperm viability; metabolism; transport; or capacitation are also involved.^ieng

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The changes induced in the relative concentrations of the different species of RNA (messenger, ribosomal, transfer and heterogenous) which regulate the metabolic behavior of tissues were studied in the endometrium of normal women and in women wearing a progesterone T IUD for 6-12 months. It was observed that the intrauterine release of progesterone is accompanied by a significant decrease in the content of total RNA (from 78 +or- 8.4 to 64 +or- 6.7 mcg/mg of protein) and in the RNA/DNA ratio (from 0.85 +or- 0.10 to 0.73 +or- 0.08). Although the protein decreased significantly (from 102 +or- 11 to 82 +or- 14 mg/g wet wt.), the DNA content and the Prot/DNA ratio were not altered. Using the affinity chromatography technique in poly(u)Sepharose it was found that the relative concentrations of RNA types were in general drastically modified: mRNA and rRNA decreased (from 2.6 +or- 0.31 to 1.28 +or- 0.15 and from 65.5 +or- 8.0 to 54.8 +or- 6.7 mcg/mg of protein respectively). tRNA content was increased (from 6.2 +or- 0.9 to 11.0 +or- 1.3) while hRNA was unchanged. The probable inhibitory effects of these changes in relation to the normal hormonal stimulation of the human endometrium, as a possible explanation of the mechanism of action of this device, are discussed.

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The binding of 3H-17-beta-estradiol to human ejaculated spermatozoa and to its subcellular structures was studied. The binding kinetics of the labeled steroid to whole spermatozoa followed a parabolic pattern. Scatchard-type plots showed the presence of high-affinity binding sites (1.56 +/- 0.23 X 10(4) per sperm cell) with an apparent Kd of 6.6 X 10(-10) M. In competition experiments testosterone was partially effective in decreasing 17-beta-estradiol binding, whereas progesterone and 17-alpha-estradiol were ineffective. Study of membrane fractions obtained from estradiol-labeled spermatozoa showed that under saturating conditions 75-84% of the bound steroid was bound to sperm membranes. Nuclear fractions obtained from estradiol-labeled spermatozoa showed only 10% of the total bound radioactivity. When isolated sperm nuclei were incubated in the presence of the purified receptor-17-beta-estradiol complex obtained from the high speed supernatant of human uterus almost no transfer of radioactivity to the nuclei was observed.

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PIP: Ribonucleic acid (RNA) metabolism of the normal and copper-treated ( Cu-T200 IUD) human endometrium was investigated. The relative concentration of total, messenger, ribosomal and transfer RNA was measured in normal and Cu-treated endometrium using the technique of affinity chromatography in polysepharose. The transition from the proli ferative to the secretory endometrium in normal women was accompanied by significant increases (p less than .05) in total RNA, messenger RNA and in ribosomal RNA. The relative proportions of bound and free messenger RNA were also modified by endometrial maturation changing from 70% bound messenger RNA in the proliferative to 83% in the secretory phase. Cu-T200 Cu release appeared to particularly affect RNA metabolism in the secretory phase. During the proliferative phase only the concentration of transfer RNA and the proportion of bound to free messenger RNA were modified by the Cu-T200. The Cu-T200 induced significant decreases (p less than .01 and p less than .05) in all RNA parameters, with the exception of the RNA/deoxyribonucleic acid ratio.^ieng

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PIP: In an effort to explain the mechanism of the increased efficacy of the copper-releasing IUD, the in vitro action of copper ions on the polysome patterns of rabbit endometrium and liver was studied. The uterine horns were extracted from female rabbits with prior sexual experience. The horns were sliced longitudinally, and the endometrium carefully scraped off with a curette. For each experiment the scrapings from 4 to 6 cornua were pooled, suspended in buffer A (.14 M sucrose containing .05 M Tris-hydrochloride, .05 M potassium chloride, .005 M magnesium chloride, 200 mcg/ml heparin and brought to pH 7.6), and divided into 5 equal fractions. To 4 of the fractions, copper (as copper chloride) was added to reach final concentrations of .15, 13, 1, and 1.5 mM, respectively. The fifth fraction served as a control with no copper added. The liver was extracted, homognized in buffer A, and treated in the same way as the endometrium. The homogenates were further adjusted, treated, centrifuged, and analyzed. Results showed that the in vitro addition of copper in concentrations as low as .15 mM produced a significant decrease in the amount of polysome aggregates that could be recovered from endometrium. Liver required concentrations of at least .3 mM to show a similar effect. For concentrations of 1.5 mM copper, the recoverable amount of the polysome fraction from liver and endometrium was 40% and 25%, respectively, of that recoverable without added copper. When the obtained polysomes were analyzed in a sucrose gradient, additions of increasing concentrations of copper induced a progressived decrease of the heavy components of the polysome patterns with a concomitant increase in the lighter components. The results which show copper as a possible dissociating agent of liver polysomes are important, but it is doubtful that liver concentrations of those required to dissociate polysomes (.3 mM) will ever be reached by the use of these IUDs. It is postulated that the impairment in polysome aggregation is part of the mechanism of the copper IUD, and it may modify both the endometrial characteristics and the blastocyst viability required for successful embryo development.^ieng

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