Alterations in epididymal sperm maturation caused by ageing.

The epididymis is an organ that performs all the biochemical changes responsible for sperm maturation. During ageing, histological alterations in the epididymis and decreased protein synthesis have been found. This might affect the sperm maturation process. The aim of this study was to determine if the changes in the epididymis during ageing might cause alterations in sperm maturation. Wistar rats of 3-4months old (young) and 18-21months old (old) were used. The testosterone concentration was determined and the epididymides were dissected and divided in three regions: caput, corpus, and cauda. The tissues were used for histological processing and sperm extraction. Testosterone concentration decreased 34% in the old animals compared to the young ones. The distribution of mannose, sialic acid, and N-acetylglucosamine in the glycocalyx of the sperm membrane of old animals was different from that of young animals. The same occurred with phosphatidylserine externalisation and protein phosphorylation at tyrosine residues. Epididymis histology in old animals showed tubular and cellular degeneration. Our results suggest that ageing affects maturational markers, likely due to alterations in the epididymis as a result of the testosterone decrease associated with ageing.

Single nucleotide polymorphisms associated with nonsyndromic cryptorchidism in Mexican patients.

Cryptorchidism is a frequent genitourinary malformation considered as an important risk factor for infertility and testicular malignancy. The aetiology of cryptorchidism is multifactorial in which certain SNPs, capable of inhibiting the development of the gubernaculum, are implicated. We analysed 16 SNPs by allelic discrimination and automated sequencing in 85 patients and 99 healthy people, with the objective to identify the association between these variants and isolated cryptorchidism. In two different patients with unilateral cryptorchidism, we found the variants rs121912556 and p.R105R of INSL3 gene in a heterozygous form associated with cryptorchidism, so we could considered them as risk factors for cryptorchidism. On the other hand, SNPs rs10421916 of INSL3 gene, as well as the variants rs1555633 and rs7325513 in the RXFP2 gene, and rs3779456 variant of the HOXA10 gene were statistically significant, when the patients and controls were compared and could be considered as protective factors since are predominantly present in controls. The genotype-phenotype correlation did not show statistical significance. With these results, we could conclude that these polymorphisms can be considered as important variants in our population and would contribute in the future knowledge of the aetiology and physiopathology of cryptorchidism.

Sexual behavior attenuates the effects of chronic stress in body weight, testes, sexual accessory glands, and plasma testosterone in male rats.

The aim of this study was to evaluate whether continuous sexual behavior could attenuate the effects of chronic stress on spermatogenesis, sexual glands, plasma testosterone and corticosterone in sexually experienced male rats. Rats were exposed to stress by immersion in cold water (ICW) daily for 20 or 50 consecutive days. Plasma testosterone and corticosterone, masculine sexual behavior, as well as the number of offspring, the epithelial area of seminiferous, prostatic and seminal glands were assessed. In stressed males, body and testicular weights decreased, male sexual behavior was disrupted, and adrenal weights increased. In males stressed for 50 days, prostate and seminal glands had lower weights compared with controls. Prostate and seminal epithelial areas also decreased in these males. Seminiferous tubules in testes from rats stressed for 20 or 50 days showed several degenerative signs, such as vacuoles in the basal epithelium, with picnotic indicia; moderate to severe exfoliation of degenerative germinal cells in the tubule lumen was also observed. In males stressed for 50 days a significant decrease in seminiferous epithelial area was observed from stages I-VIII, regardless of copulation. The litters from females that copulated with males stressed for 50 days decreased significantly. Chronic stress caused increase in plasma levels of corticosterone, which were higher in males stressed for 20 days than in males stressed for 50 days. Testosterone decreased in stressed males and it was lower in males stressed for 50 days. In stressed males allowed to copulate, body and testicular weights were similar to controls. Adrenal, seminal glands, and prostate weights, as well as epithelial areas of males stressed for 50 days allowed to copulate were also similar to controls. Corticosterone was lower than in males stressed for 50 days, but still higher than in controls. Testosterone in males stressed for 50 days and allowed to copulate was higher than in stressed males not allowed to copulate and control males without copulation, but still lower than in control copulating males. These results show that chronic stress causes germ cell loss in testes and a decrease in prostate and seminal epithelium, possibly as a result of testosterone decrease, affecting fertility. Continuous copulation can attenuate the effects of stress on testosterone levels and on the epithelial area in male sexual glands, but not on the seminiferous epithelium after 50 days of stress.

Infertility in rats subjected to genitofemoral nerve section is not associated with testicular damage.

This work was aimed at assessing the relationship between testicular ascent and infertility induced by genitofemoral nerve (GFN) section in rats. Eighteen male rats were assigned to three experimental groups as follows: (i) Group SGFN was subjected to surgical section of genitofemoral nerve; (ii) Group Sham; (iii) Control group. The GFN was cut at puberty (28D), and the contralateral testis removed at 90D, with fertility tests at 120D. At 150D, maturity index, epithelial area and histopathological index of seminiferous tubules of all rats were determined and statistically compared between superior and inferior testicle poles, and between groups. There were no differences in testicular parameters, sperm morphology or sperm concentrations (P > 0.05). Section of NGF interfered with fertility (58.3 ± 15.4 in SGFN versus 83.3 ± 10.5 in Sham) and litter size (6.2 ± 1.1 in SGFN versus 10.7 ± 1.4 in Sham). Cremaster of SGFN group showed early neuropathy. The GFN section induced partial testicular ascent and diminished fertility without damage on testicular morphology or spermatic parameters, because, cremaster could affect the contractibility and ejaculation mechanisms in which it participates. The study of the damage on cremaster induced by an injury on GFN could have an overview of the mechanisms inherent in the testicular ascent induced by this iatrogenic alteration and their potential risks on fertility.

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.

Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.

Testosterone, androstenedione, and 5alpha-dihydrotestosterone on male sexual behavior and penile spines in the hamster.

The expression of masculine sexual behavior (MSB) in male hamsters is optimally stimulated by aromatizable androgens like androstenedione (AD) and testosterone (T), while the non-aromatizable androgen, 5alpha-dihydrotestosterone (DHT), exerting potent androgenic peripheral effects, only in high doses maintains MSB after castration. No data exist on the ability of these androgens to restore long intromissions after castration. In this study, AD, T, and DHT were administered to four-week gonadectomized, sexually experienced male hamsters, for three weeks, in doses of 25 microg/day or up to 1000 microg/day to compare their potency in restoring MSB, penile size, and penile spines growth. Plasma levels of these steroids and the metabolites estrone and estradiol, were determined at the end of the treatment period. Gonadectomy completely suppressed MSB and induced a regression of penile spines. AD was more potent than T in restoring MSB, ejaculatory behavior being displayed by most castrated subjects with a lower dose of AD (50 microg/day) than of T (300 microg/day), and long intromissions being shown by all AD-treated castrated hamsters but only by 20% of T-treated ones, when doses of 1000 microg/day were given. DHT did not stimulate any copulatory response. The three androgens, even at the lowest dose, partially stimulated penis and penile epithelium growth, DHT showing the highest potency. Treatment of castrated hamsters with AD (50 microg/day), restored steroid levels to similar values as those of intact animals. These results show that AD and T restored MSB even with a partial stimulation of penile spines growth, AD being more potent than T. In contrast, DHT did not restore MSB in the hamster in spite of its peripheral androgenic potency.

Immunoreactive vasoactive intestinal polypeptide and vasopressin cells after a protein malnutrition diet in the suprachiasmatic nucleus of the rat.

The aim of the present study was to evaluate the effects of prenatal and postnatal protein deprivation on the morphology and density of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) immunoreactive neurons in the suprachiasmatic nucleus (SCN) of young rats. Female Wistar rats were fed either 6% (malnourished group) or 25% (control group) casein diet five weeks before conception, during gestation and lactation. After weaning, the pups were maintained on the same diet until sacrificed at 30 days of age. The major and minor axes, somatic area and the density of VP- and VIP-immunoreactive neurons were evaluated in the middle sections of the SCN. The present study shows that chronic protein malnutrition (ChPM) in VP neurons induces a significant decrease in number of cells (-31%,) and a significant increase in major and minor axes and somatic area (+12.2%, +21.1% and +15.0%, respectively). The VIP cells showed a significant decrease in cellular density (-41.5%) and a significant increase in minor axis (+13.5%) and somatic area (+10.1%). Our findings suggest that ChPM induces abnormalities in the density and morphology of the soma of VP and VIP neurons. These alterations may be a morphological substrate underlying circadian alterations previously observed in malnourished rats.

Identification of serotoninergic system components in stallion sperm.

Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several reproductive processes, i.e., it enhances hyperactivation, motility, and promotes the acrosome reaction in mammalian sperm, but it has not been described in the stallion sperm yet. Therefore, using both immunofluorescence and western blot techniques, we searched for and found some serotonin markers such as 5-HT, 5-HT1B, 5-HT2A, 5-HT3 receptors, both TPH1 and MAOA enzymes, and serotonin transporter (5-HTT) in stallion sperm. In addition, we found a non-neuroendocrine cell, V-MAT1 transporter, which has not been previously reported in mammalian sperm. Our results suggest that serotoninergic system is present in stallion sperm, which could be a pharmacological target to increase the conception rates in domestic horses.

Epithelial inclusion cysts following radial keratotomy.

A 26-year old woman underwent radial keratotomy combined with concentric trephination (6.5 and 8 mm) six months prior to development of multiple epithelial inclusion cysts. A biopsy specimen from one enlarging cyst showed an absence of scarring in addition to the retention of epithelium within a radial incision wound. Adjacent keratocytes were abnormal and seemed to be undergoing degenerative changes. To our knowledge, this is the first report of a failure in corneal wound healing associated with microcystic degeneration of the epithelium following radial keratotomy.

A modified artificial anterior chamber for use in refractive keratoplasty.

We devised a modified artificial anterior chamber that enables Barraquer microkeratome keratectomies on corneas preserved in McCarey-Kaufman medium. It consists of a hydraulic system, which creates a positive pressure of 60 mm Hg or more. An added rotating ring serves as a base to the microkeratome's tracks and varying diameters of lamellar sections can be obtained. This device allows the surgeon to use readily available corneas with scleral rims preserved in McCarey-Kaufman medium rather than whole eyes to obtain precise lamellar sections.

Variations in corneal wound healing after radial keratotomy: possible insights into mechanisms of clinical complications and refractive effects.

Ultrastructural and histopathologic analysis was performed on three human corneal specimens for variable and complicated refractive outcomes 1-2 years after radial keratotomy. Specimens were obtained immediately postsurgery after microkeratome resection with homoplastic lamellar keratoplasty (two cases) and penetrating keratoplasty (one case) for correction of glare, severe astigmatism, overcorrection, and/or double vision. All three cases showed variability of wound healing and delayed corneal wound healing sites; epithelial retention cysts, and/or absence of stromal scar collagen that was not dependent on the length of time after surgery. Two of the three radial keratotomy specimens also contained extensive duplication of the superficial corneal epithelial basal lamina. When present, the thickened basal lamina (3-6 microns in thickness) was seen between all incisions evaluated and appeared to extend from the central optical zone out to the periphery of the lamellar button. The one full-thickness keratoplasty specimen showed focal loss of underlying endothelial cells with occasional migrating cells seen by scanning electron microscopy. These data support previous findings that delayed corneal wound healing with epithelial retention cysts remains the most common histopathologic alteration after radial keratotomy. The effects of variations and delay in wound healing between individuals could explain the lack of predictability of refractive outcome and continuing refractive instability in long-term follow-up after single or repeat radial keratotomy surgeries.

[Comparison of 2 types of experimental transplant in the treatment of lethal short bowel syndrome]. / Comparación de dos tipos de trasplante experimental en el tratamiento del intestino corto letal.

OBJECTIVE: To evaluate the functional response, morbidity and histostructural changes in rats enterectomized and without cecum using two types of syngenic enteral transplants. MATERIAL AND METHODS: Controlled randomized surgical-therapeutic trial. Four groups of male Lewis rats 8-10 weeks old underwent the following procedures: 1. Lethal enteral resection (n = 10). 2. Lethal enteral resection + total yeyuno-ileal transplant (n = 28). 3. Lethal enteral resection + distal segmentary of 40% and cecum transplant (n = 32). 4. Control group (n = 10). RESULTS: 11% of the transplanted animals died due to technical failures; both transplanted groups had a similar proportion of late complications, mostly enteral obstruction. A persistent diarrhea was observed in 20% of the yeyuno-ileal transplanted group, but no significant differences were found between the two groups concerning survival, weight gain, protein and triglycerides serum levels, and a maltose absorption test; villus and crypt hypertrophy was observed in both grafts. The enteral graft integration was followed by structural changes similar to those found in intestinal remnants on deficit conditions after enteral resection. CONCLUSION: The bowel distal segmentary transplant with ileocecal valve and cecum may be a good option in cases of irreversible enteral failure, as the functional response and morbidity are similar to those found with the standard total transplant.

[Enteral resection in the nursing rabbit (an experimental study)]. / Resección enteral en el conejo lactante. (Estudio experimental).

The response to enteral resection in human newborns and nursing babies depends on the site and magnitude of the resection: usually these patients have a satisfactory recovery although long term supportive measures are required in order to prevent nutritional and digestive complications. This study assessed the post-surgical response of six groups of nursing rabbits with 0, 40, 50, 60, 75 and 90% selective resection of the small intestine. A greater number of surgical complications was observed in those cases with more manipulation and greater resection. Villi hypertrophic was found at the distal extremes of the enteral remnants only with 40% jejunal resection and 75 and 90% jejunum-ileum resections. None of the groups showed growth interruption. Only in rabbits with resections greater than 60% a significant decrease in weight was found. Forty and sixty percent enteral resections, mainly proximal and distal respectively, did not yield weight or length deficits as compared with the control group.

[The effects of early and late weaning on the development of the small intestine in the rat: a light-microscopic morphometric study]. / Efectos del destete precoz y tardío sobre el desarrollo del intestino delgado de la rata: un estudio morfométrico a nivel de microscopía optica.

UNLABELLED: The small intestine of the rat shows morphologic and enzymatic changes that are associated with the weaning and may be alternated by the early weaning, however, the morphometric criteria have been disregarded. MATERIAL AND METHODS: In this study, the effects of precocious weaning (15 days) and prolonged weaning (32 days), on the size and number of villi; and crypts of small intestine, were analyzed in rats from 16 to 70 days of age. RESULTS: Precocious weaning increased the size of villi, depth and number of crypts in the duodenum and jejunum, while the number of villi decreased. Pups nursed up to 32 days showed no alterations in the analyzed parameters. However, the ileum showed no alterations with the precocious weaning or prolonged. CONCLUSIONS: These data support the concept of an intrinsic biologic program as control of intestinal development while the change of diet seem to have a modifying role in duodenum and jejunum.

Effect of pyrimethamine treatment on male rat testicular cell population development.

Pyrimethamine (PYR) is a drug used in the treatment of newborn with congenital Toxoplasmosis. Even when PYR is highly specific against parasites, it may provoke neutropenia in the patients apart from other affectations, conditions that usually justify its suspension. Moreover, medication against congenital toxoplasmosis coincides with the proliferation stage of Sertoli and germ cells. Although, there are several reports on the effect of this drug on mature testes, records of its effects on the testes of young individuals yet in the process of growth are still lacking. This work was aimed to study the effects of in vivo administration of PYR in the first 21 days of life of male rat pups by evaluating their testicular alterations and its long-term sequels on fertility. Through the determination of the levels of seminiferous epithelium maturity, apoptotic index and cell proliferation index at 7, 14, 35 and 90 days post-natal using immunocytochemical studies. The fertility of the treated rats was evaluated at 90 days. PYR-treated animals were found to undergo some kind of delays in seminiferous epithelium maturity, decreased cell proliferation index and an increase in apoptosis when compared with the control (p < 0.05). Epididymal sperm counts were also affected (p < 0.05). The application of folic acid (FA) in newborns treated with PYR decreased the severity of the problem (p < 0.05). This study provides strong evidence that the effect of PYR on testicular development is specific. It reinforces the importance of FA application in neonates treated with PYR to prevent the effect of the later on spermatogenesis.

Macropinocytosis of the PDGF ß-receptor promotes fibroblast transformation by H-RasG12V.

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor ß-receptor (PDGFRß) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRß activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRß macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRß activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.

Maternal protein restriction in pregnancy and/or lactation affects seminiferous tubule organization in male rat offspring.

Maternal protein restriction (MPR) during pregnancy impaired the reproduction of male offspring. We investigated, during the first wave of spermatogenesis, whether MPR exerts deleterious effects on germ cell proliferation and differentiation, as well as androgen receptor (AR) protein expression, which was used as a marker for Sertoli cell (SC) maturation. At the beginning of pregnancy (day 0), dams were fed a control diet (C: 20% casein) or a restricted isocaloric diet (R: 10% casein). After birth, four groups were established: CC, RR, CR and RC (first letter diet during pregnancy and second during lactation). Male offspring were studied at postnatal days 14, 21 and 36. At birth, pup body weight was unchanged. Body weight and testis weight were reduced in RR and CR groups at all ages evaluated. MPR delayed the germinal epithelium development at all ages evaluated. On performing Western blot and immunohistochemistry, AR expression was found to be lower in the three restricted groups. The results suggest that MPR during pregnancy and/or lactation delays SC maturation and germ cell differentiation, and affects intratubular organization. These changes might be responsible for the lower fertility rate at older ages.

Toxicity of the crude oil water-soluble fraction and kaolin-adsorbed crude oil on Daphnia magna (Crustacea: Anomopoda).

Crude oil is a complex mixture of hydrocarbons entering aquatic environments from accidental or normal marine and transportation activities. Toxicologic crude oil analysis is usually performed on the basis of the water-soluble fraction. However, this yields only a partial estimate of the damage caused by these contaminants because a substantial hydrophobic amount can be adsorbed onto suspended solids (biotic and abiotic), which directly affects filter-feeding species and permits bioaccumulation through trophic relationships. This study determined the acute toxic damage sustained after 48 hours caused by seven types of crude oil from Tabasco, Mexico on the cladoceran Daphnia magna. Comparisons were documented based on the responses of D. magna from application of the water-soluble fraction and exposure to entire crude oil samples adsorbed on kaolin clay. Oil-sorbed kaolin was more toxic than the water-soluble fraction in acute exposure. This confirms that tests of the water-soluble fraction tend to underestimate the toxic damage that can be produced in natural environments. Furthermore, chronic toxicity (21 days) was evaluated for crude oil samples adsorbed on kaolin at sublethal concentrations as established from Application Factors (AF) criteria. Results showed that in most cases, it is impossible to predict safe concentrations on the basis of LC(50) values because samples with lower acute toxicity exercised a greater influence on D. magna reproduction and survival when subjected to chronic exposure.