Copulatory mechanism and functional morphology of genitalia and anal horn of the scorpionfly Cerapanorpa dubia (Mecoptera: Panorpidae).

The scorpionfly genus Cerapanorpa is characterized by the male possessing a single finger-like anal horn on the posterior portion of tergite VI. However, the functional morphology of this anal horn and the genitalia have not been studied to date. Herein, we investigated the functional morphology of the genitalia and the nongenital structures of the scorpionfly Cerapanorpa dubia by observing the mating process and dissecting the freeze-fixated pairs in copula to reveal the copulatory mechanism. The male C. dubia provides a solid salivary mass to the female as a nuptial gift prior to copulation. When the female starts to feed on the gift, the male uses his notal organ and complex genital structures to control the closest wing and genitalia of the female to establish a V-shaped mating position. In the maintenance phase of copulation, the male uses his anal horn in cooperation with the basally-constricted abdominal segment VII to clamp female abdominal segment VIII. The male hypovalves grasp female cerci, and move up and down rhythmically. The paired parameres clasp both sides of female tergite IX. The basal processes on male gonostyli grip the pleural membranes of the female genital chamber. In the sperm transfer phase, the male aedeagus directly couples with the female medigynium to transmit sperm by connecting his phallotreme to the female's copulatory pore. The evolution of the male complex grasping structures in Panorpidae is also briefly discussed.

Influence of neoadjuvant chemoradiotherapy on the anal sphincter: ultrastructural damage may be critical.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) in addition to the curative surgery has been the first of treatment for local advanced rectal cancer because of its benefits in local recurrence and sphincter-saving. However, its side effects on anorectal function have been recognized. The histopathological changes on internal anal sphincter (IAS) have been reported, but ultrastructure changes of external anal sphincter (EAS) are unknown. The aim of this study is to detect the alterations on the gross morphology of IAS and ultrastructure of EAS after nCRT. METHODS: We collected 34 anal canal specimens of patients undergoing abdominoperineal resection (APR) prospectively. The length and thickness of IAS were measured with vernier caliper. The EAS was dissected for observation with transmission electron microscope (TEM). RESULTS: Ten patients received nCRT (nCRT group) before surgery and 24 underwent APR directly (control group). The length and thickness of IAS in nCRT group were 22.68 ± 3.56 and 5.39 ± 0.74 mm, respectively. These parameters were 21.28 ± 3.62 and 5.35 ± 1.12 mm in control group, respectively. There were no significant differences in the length and thickness of IAS between the two groups (P>0.05). In nCRT group, the sarcomere and myofibril were arranged disorderly and parts of them that were filled with collagenous fiber, triads, and mitochondria were destroyed severely and the glycogenosome also distributed disorderly. Such alterations of EAS did not occur in control group. CONCLUSIONS: The nCRT cannot change the gross morphology of IAS, while it induces serious damages to the ultrastructures of EAS which may adversely affect the anorectal function.

Nerves supplying the internal anal sphincter: an immunohistochemical study using donated elderly cadavers.

PURPOSE: Nerves serving the internal anal sphincter (NIAS) have been described as the lower rectal branches of the pelvic autonomic nerve plexus. However, their topographical anatomy and fiber components have remained unclear. METHODS: Using histological sections from ten elderly donated cadavers, we investigated the topographical anatomy and composite fibers of the NIAS using immunohistochemistry for S100 protein, neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). RESULTS: At the 2-3 o'clock position in the lower rectum, the NIAS originated from nerves at the posterolateral corner of the prostate in males or in the lower paracolpium in females. The nerves ran inferiorly along the internal aspect of the levator ani muscle, and joined branches of the myenteric plexus at a level slightly above the epithelial junction. The NIAS contained both nNOS-positive parasympathetic nerve fibers and TH-positive sympathetic fibers, but VIP-positive fibers were few in number. CONCLUSIONS: The origin of the NIAS at the posterolateral corner of the prostate as well as in the lower paracolpium might be sacrificed or damaged during radical prostatectomy or tension-free vaginal tape insertion. Low anterior resection of rectal cancer will most likely render damage to the NIAS because of its intersphincteric course. Although the nerve composition of the NIAS is characterized by a higher proportion of sympathetic nerve fibers than the myenteric plexus in the large intestine, their role is unclear. However, evaluation of sphincteric function after surgery would appear to be difficult because of the complex control mechanism independent of nerve supply.

Spatiotemporal expression of fibroblast growth factor 10 in human hindgut and anorectal development.

As fibroblast growth factor 10 (FGF-10) gene expression may have a role in anorectal duct formation, this study aimed to assess the spatiotemporal expression pattern of FGF-10 during development of the rectum and hindgut in human embryos. FGF-10 expression was evaluated in human embryos (n = 85) at 3-8 weeks of gestation after immunohistochemical evaluation using antibodies specific for FGF-10. From weeks 4 to 7 of gestation, FGF-10 expression was observed primarily in the apical epithelium of the dorsal urorectal septum, the cloacal membrane (CM) and the hindgut. Following CM rupture (week 7), the epithelium of the anal canal was negative for FGF-10; however, it was present within the urothelium through week 7. FGF-10 expression during the development of the human hindgut and anorectum suggests that it may play a role in hindgut and anorectal morphogenesis.

Cytochemistry of sialoglycoconjugates and lysozyme in canine anal glands as studied by electron microscopic methods.

The distribution of sialoglycoconjugates and lysozyme in the secretory cells of canine anal glands was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry and immunocytochemistry. Sialic acids were predominantly present in the secretory granules, Golgi bodies, surface coat of the plasma membrane and luminal secretions. In addition, within these structures, the secretory granules, Golgi bodies and luminal secretions exhibited high levels of sialoglycoconjugates that terminated in Siaα2-6Gal/GalNAc or Siaα2-3Galß1-4GlcNAc. In the secretory cells, reactive gold particles representing lysozyme were mainly detectable in the secretory granules and Golgi bodies. Sialic acids possess diverging functional properties, whereas lysozyme contributes to the non-specific defense against microorganisms. Therefore, their presence and secretion are suggestive of protective effects of both secretory products at the anal mucosa.

[Anorectal manometry and ultrasound in the study of patients with fecal incontinence]. / Ecografía anorrectal y manometría en el estudio de pacientes con incontinencia fecal.

INTRODUCTION: Fecal incontinence is a complex disease that affects the quality of life of patients suffering from it. Anorectal manometry and endoanal ultrasound 360 grades are included among the studies used. There are no clear agreement about the relevance and usefulness of this studies and their role as apart of diagnostic procedures. OBJECTIVE: To describe the relationship between the clinical severity score in patients with fecal incontinence and the findings of anorectal manometry and endoanal ultrasound. Another objective of this study is to describe the degree of agreement of both methods of study. MATERIAL AND METHODS: The study population includes 74 patients who underwent endoanal ultrasonography and anorectal manometry for fecal incontinence. The presence or absence of ultrasound injury, the pressures obtained by anorectal manometry and its relationship with clinical severity of patients were described. An analysis of the degree of agreement between both methods was performed. RESULTS: The mean age of patients was 53 years old (range 19-84 years). Pressures of anorectal manometry were reduced in 59 patients (79.7%) and normal in 15 (20.3%). The assessment of the degree of agreement or concordance between ultrasound and anorectal manometry yielded a kappa coefficient of 0.25 (acceptable) (P = 0.0001). CONCLUSION: There is a level of acceptable agreement between endoanal ultrasound findings and anorectal manometry when patients with fecal incontinence are evaluated.

Morphology of the epithelium of the lower rectum and the anal canal in the adult human.

The anal canal is an important body part clinically. However, there is no agreement about the epithelium of the anal canal, the anal transitional zone (ATZ) epithelium in particular. The aim of this study is to clarify the structure of the epithelium of the human lower rectum and anal canal. Intact rectum and anus obtained from patients who underwent surgery for rectal carcinoma were examined by light and scanning electron microscopy (LM and SEM). By LM, three types of epithelium were observed in the anal canal: simple columnar epithelium, stratified squamous epithelium, and stratified columnar epithelium. The lower rectum was composed of simple columnar epithelium. SEM findings showed stratified squamous epithelium that consisted of squamous cells with microridges, changing to simple columnar epithelium consisting of columnar cells with short microvilli at the anorectal line. LM and SEM observations in a one-to-one ratio revealed that the area of stratified columnar epithelium based on LM corresponded to the anal crypt and sinus. In conclusion, the epithelium of the human anal canal was fundamentally composed of simple columnar epithelium and stratified squamous epithelium. We found no evidence of the ATZ.

Accuracy of the digital anal examination in women with fecal incontinence.

INTRODUCTION AND HYPOTHESIS: This study aims to determine the accuracy of digital rectal examination (DRE) to detect anal sphincter defects when compared to endoanal ultrasound (US) in women with fecal incontinence (FI). METHODS: Seventy-four patients identified by retrospective chart review who presented with complaints of bothersome FI who underwent endoanal US are the subjects of this analysis. Sensitivity, specificity, and positive and negative likelihood ratios were calculated for the ability of the DRE to detect anal sphincter defects. RESULTS: Anal sphincter defect was suspected on DRE in 75%. At endoanal US, external sphincter defects were noted in all three segments in 41% (complete defect) while partial defects were noted in 30%. DRE demonstrated a sensitivity of 82%, specificity of 32%, +likelihood ratio 1.2 (95% confidence interval (CI), 0.95-1.16) and -likelihood ratio of 0.6 (95% CI, 0.2-1.24) for detecting a complete EAS defect on endoanal US. CONCLUSION: DRE has poor specificity for detecting anal sphincter defects seen on endoanal US.

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter.

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα(+)), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα(+) cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα(+) cells and neuronal nitric-oxide-synthase-positive (nNOS(+)) nerve fibers in the IAS of wild-type (WT) and W/W ( v ) mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα(+) cells (PDGFRα(+)-IM). Other populations of PDGFRα(+) cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ( v ) IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα(+)-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ( v ) IAS. Minimum surface distance analysis revealed that nNOS(+) nerve fibers were closely aligned with both ICC-IM and PDGFRα(+)-IM. An even closer association was seen between ICC-IM and PDGFRα(+)-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα(+)-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice.

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin alpha6, and thus represent a putative anal stem cell population.

Effects of ovarian failure on submucosal collagen and blood vessels of the anal canal in postmenopausal women.

BACKGROUND: Estrogen and progesterone receptors are expressed in the anal canal. Fecal control deteriorates after menopause. This phenomenon is related to decreased circulating levels of estrogen and progesterone due to ovarian failure at menopause. AIM OF WORK: To study the effects of estrogen and progesterone on inflammatory cells, submucosal collagen fibers, and vascular plexus of the anal canal of postmenopausal women. SUBJECTS AND METHODS: Experiments were performed on samples of anorectal tissue obtained from 40 women, 19 menstruating (group I), and 21 postmenopausal women (group II). Investigations included immunohistochemistry of estrogen and progesterone receptors and CD34. RESULTS: In negative estrogen receptors (ER) and progesterone receptors (PR), inflammatory cells, submucosal blood vessels, collagen type I were nonsignificantly changed in postmenopausal women relative to menstruating women (P > 0.05) whereas, in positive ER and PR, inflammatory cells and collagen I were significantly increased and submucosal blood vessels were significantly decreased in postmenopausal women relative to menstruating women (P < 0.05). CONCLUSION: Estrogen and progesterone, in menstruating women, produce beneficial effects by decreasing incidence of inflammation and increasing anal canal submucosal blood vessels number and collagen types I, thus both hormones have a positive effect on anal compliance and pressure.

Role of musculature during defecation in a particle-feeding arachnid, Archegozetes longisetosus (Acari, Oribatida).

Most extant Chelicerata are characterized by external digestion and the ingestion of fluid food. Exceptions include the marine taxa, most Opiliones, and the mite groups Opilioacarida (Parasitiformes) and Sarcoptiformes (Acariformes), which ingest particulate food. This leads to different physiological and morphological adaptations for food processing, including the production and extrusion of solid fecal pellets, which are rather large in sarcoptiform mites. Few studies have investigated the defecation of such large fecal pellets, and available information is contradictory. We use a combination of non invasive microscopical techniques and in vivo examination to investigate the complex functional morphology of the anal region of the oribatid mite Archegozetes longisetosus Aoki. The opening of the anus is at least initiated by indirect muscular action via an increase of hemolymph pressure, through the action of dorsoventral muscles (dvm). Extrusion of the fecal pellet is accomplished by the prerectal muscle collar, with full opening of the anus and rotation and bowing of the plates probably resulting from pressure of the pellet. The sequential nature of these actions was demonstrated by many observations in which the anus opened only partially; these were concomitant with dmv contraction but pellets were not being extruded. All muscles directly connected to the anal and adanal regions assist in keeping the anus closed; they are antagonists to hydrostatic forces that are necessary for normal activity. Based on the literature, no obvious similarities were noted with defecation musculature in other particle-feeding chelicerates, but most muscles can be homologized with those of more specialized oribatid mites. The function of the outer anal muscles has been modified in both Euphthiracaridae and Brachypylina to assist in providing general hemolymph pressure.

Development of the anal vesicle, salivary glands and gut in the egg-larval parasitoid Chelonus inanitus: tools to take up nutrients and to manipulate the host?

Larvae of endoparasitoids undergo extensive morphological changes and often have special features to allow their development inside the host. We present the first detailed study on the development of the anal vesicle and the gut. The analyses reveal that the anal vesicle is first seen on the dorsal side of the abdomen as an internal structure covered by a membrane. The morphology of the abdomen then changes intensively: new segments are formed and the anal vesicle develops from a crest of large cells to a protrusion. Towards the end of the first instar, the anal vesicle is fully evaginated and no longer covered by a membrane; the large epithelial cells have microvilli on their apical side which suggests uptake of nutrients from the host's haemolymph. When the larva has moulted to the second instar, the ultrastructure of the anal vesicle begins to change and shows signs of degeneration. In this stage the epithelium of the midgut is fully developed and has a brush border which suggests that nutrient uptake occurs now primarily through the midgut. The anal vesicle then degenerates completely. The salivary glands are prominent already in first instar larvae and appear to produce and release a host regulatory 212 kD protein.

Ultrastructural organization of the anal organs in the anal capsule of Craterostigmus tasmanianus Pocock, 1902 (Chilopoda, Craterostigmomorpha).

We describe the ultrastructural organization of the anal organs of Craterostigmus tasmanianus, which are located on the ventral side of the bivalvular anal capsule. Each part of the capsule bears four pore fields with several anal pores. The pores lead into a pore canal, which is surrounded by the single-layered epithelium of the anal organs. Each anal organ is composed of four different cell types: transporting cells of the main epithelium, junctional cells, isolated epidermal glands, and the cells forming the pore canal. The transporting cells exhibit infoldings of the outer cell membranes, forming a basal labyrinth and a poorly developed apical complex. The cells are covered by a specialized cuticle with a widened subcuticular layer. Only the cuticle of the main epithelium is covered by a mucous layer, secreted by the epidermal glands. The ultrastructural organization of the anal organ is comparable to the coxal and anal organs of other pleurostigmophoran Chilopoda. It is likely that the coxal and anal organs of the Pleurostigmophora are homologous, due to their identical ultrastructural organization. Differences concerning the location on the trunk of Pleurostigmophora are not sufficient to reject a hypothesis of homology. Anal organs are found not only in Craterostigmomorpha, but also in most adult Geophilomorpha, and in larvae and most adults of Lithobiomorpha. The anal organs of C. tasmanianus are thought to play an important role in the uptake of atmospheric water. J. Morphol.

¿Es útil la ecografía endoanal de rutina en las fístulas perianales? / Is routine endoanal ultrasound useful in anal fistulas?

Objetivo: evaluar la efectividad de la ecografía endoanal contrastada con inyección de agua oxigenada para establecer la topografía fistulosa (trayecto y localización del orificio interno) y valorar la conveniencia de esta exploración en la supuración anal o perianal cuando es realizada por un cirujano general dedicado a coloproctología y entrenado en esta técnica. Pacientes: se hizo ecografía endoanal a 103 pacientes con supuración anal o perianal. Se excluyeron 20 pacientes del estudio: 9 tenían el orificio externo de la fístula cerrado y 11 presentaban abscesos de origen criptoglandular. Todas las ecografías fueron hechas por el mismo explorador utilizando un ecógrafo B&K con sonda rotatoria de 7 MHz. La exploración consistía en identificar los tres planos ecográficos del canal anal, inyectar agua oxigenada por el orificio externo y repetir el procedimiento. Resultados: de los 83 pacientes incluidos, 11 resultaron ser sinus y 72 fístulas perianales. De estas se localizó el trayecto principal en todas, identificándose 24 interesfinterianas (33,33%), 33 transesfinterianas (45,83%), 3 supraesfinterianas (4,17%) y 12 extraesfinterianas (16,67%). Se localizó el orificio interno en 69 (95,83%). Conclusiones: la ecografía endoanal realzada con agua oxigenada es un método efectivo para visualizar el trayecto y el orificio interno de las fístulas perianales. Creemos que resulta de gran utilidad en todos los casos de supuración anal o perianal para localizar colecciones, reconocer sinus perianales, conocer el estado de los esfínteres y planificar la cirugía

Objective: to evaluate the effectiveness of endoanal ultrasound with hydrogen peroxide enhancement in the assessment of anal fistula (tract and internal opening), and to value the utility of this examination for anal or perianal suppuration when performed by a colorectal surgeon trained in this technique. Patients: endoanal ultrasound was performed in 103 patients with anal or perianal suppuration. Twenty patients were excluded: 9 had the external opening closed, and 11 had cryptoglandular abscesses. All ultrasound scans were performed by the same explorer using a B&K Diagnostic Ultrasound System with a 7 MHz endoprobe. The examination was based on the identification of the three anal planes, then hydrogen peroxide was infused and the procedure was repeated. Results: out of 83 patients included, 11 had a perianal sinus and 72 an anal fistula. In all fistulas the main tract was found: 24 were inter-sphinteric (33.33%), 33 trans-sphincteric (45.83%), 3 supra-sphincteric (4.17%), and 12 extra-sphincteric (16.67%). An internal opening was identified in 69 (95.83%). Conclusions: endoanal ultrasound with hydrogen peroxide enhancement is an effective examination to visualize fistulous tracts and internal openings. We think it is highly useful for anal or perianal suppuration to identify abscesses, to recognize a perianal sinus, to check the sphincteric condition, and to plan subsequent surgery

Fine structure of the anal tonsilliar epithelium in the laboratory shrew (Suncus murinus).

The epithelium of the anal tonsil of the laboratory shrew was studied by scanning and transmission electron microscopy, with particular attention focused on the structure of the epithelium lining the anal tonsillar crypt. The tonsillar crypt surface is lined by two kinds of epithelia: squamous epithelium, which is located mainly at the neck of the crypt and includes keratohyalin granules in the superficial layer, and reticular epithelium, which is invaded by many immigrating cells and has several micropores immigrating cells to pass through. In addition, basal granulated cells are present in the basal layer. These results suggest that the reticular epithelium of the anal tonsil belongs to the well-developed gut-associated lymphoid tissue (GALT) in the alimentary canal. It represents a specialized and important compartment in immunological function, similarly to the palatine tonsils of other mammals, and has as yet unknown roles in digestion.

Distribution of interstitial cells of Cajal in tunica muscularis of the canine rectoanal region.

Electrical and mechanical activity of the circular muscle layer in the rectoanal region of the gastrointestinal tract undergoes considerable changes in the site of dominant pacemaking activity, frequency, and waveform shape. The present study was performed to determine whether changes in the structural organization of the circular layer or in the density, distribution, and ultrastructure of interstitial cells of Cajal (ICC) could account for this heterogeneity in electrical and mechanical activities. Light microscopy revealed that the structural organization of the circular muscle layer underwent dramatic morphological changes, from a tightly packed layer with poorly defined septa in the proximal rectum to one of discrete muscle bundles separated by large septae in the internal anal sphincter. Kit immunohistochemistry revealed a dense network of ICC along the submucosal and myenteric borders in the rectum, whereas in the internal anal sphincter, ICC were located along the periphery of muscle bundles within the circular layer. Changes in electrical activity within the circular muscle layer can be partially explained by changes in the structure of the muscle layer and changes in the distribution of ICC in the rectoanal region of the gastrointestinal tract.

Alteration of anal sphincter morphology following vaginal delivery revealed by multiplanar anal endosonography.

OBJECTIVE: To assess morphologic change in the anal sphincters in the absence of endosonographic evidence of trauma after vaginal delivery. DESIGN: Prospective observational study. SETTING: District general hospital. POPULATION: Consecutively booked nulliparous pregnant women attending antenatal clinic. METHODS: All women were examined using three-dimensional anal endosonography, simple manometry and had questionnaire assessment of incontinence before and after delivery. MAIN OUTCOME MEASURES: Components of the anal canal were measured in the axial, sagittal and coronal planes and paired pre- and post-delivery examinations were compared. Any changes were related to changes in continence and anal canal manometry. RESULTS: Twenty-two women had a vaginal delivery and no endosonographic evidence of perineal trauma after delivery. After delivery, there was significant shortening of the length of the anterior external anal sphincter [EAS] (mean 21.7 vs 20.5 mm, P = 0.02) when measured in the sagittal plane, which increased in anterior angulation with respect to the axis of the anal canal (10 degrees vs 13.8 degrees, P = 0.03). In the axial plane, no change was seen in the thickness of any of the sphincter components after delivery. None of these morphologic changes correlated with changes in manometry or continence score. CONCLUSIONS: Anal sphincter morphology changes after an otherwise atraumatic vaginal delivery. This change does not correlate with any functional symptoms.

[Regeneration of striated muscular tissue of the external rectal sphincter in the rat]. / Regeneratsiia poperechnopolosatoi myshechoi tkani naruzhnogo sfinktera priamoi kishki krysy.

The reparative regeneration of striated muscular tissue of the external rectal sphincter was studied in albino rat following its injury caused by rectal stretching. It was shown that despite some peculiar features, the regeneration developed in accordance with histogenetic determination. Along with the death of some muscle fibers, some others were activated with the formation of undifferentiated elements. Proliferation of appearing myoblasts was followed by their differentiation and integration with the formation of new muscle fibers.