Three-dimensional architecture of the myosalpinx in the mare as revealed by scanning electron microscopy.код для вставкиСкачать
THE ANATOMICAL RECORD 267:235–241 (2002) Three-Dimensional Architecture of the Myosalpinx in the Mare as Revealed by Scanning Electron Microscopy ANTONINO GERMANÀ,1 ROSA CASSATA,2 SANTO CRISTARELLA,2 AURELIO SCIRPO,2 AND UGO MUGLIA1* 1 Department of Morphology, Biochemistry, Physiology, and Animal Productions, Morphology Section, Veterinary Faculty, University of Messina, Messina, Italy 2 Department of Surgery, Physiopathology, and Animal Reproduction Clinic, Veterinary Faculty, University of Messina, Messina, Italy ABSTRACT The three-dimensional architecture of the myosalpinx in the mare was investigated by means of scanning electron microscopy (SEM) after removal of interstitial connective tissue with NaOH digestion. In the extramural portion of the tubo-uterine junction (TUJ), isthmus, and ampulla, the myosalpinx architecture is represented by a unique muscular structure which runs from the mesosalpinx to the base of the inner mucous folds. This unique muscular structure consists mainly of bundles of muscular fibers independent of one another, which show a multiple spatial arrangement and form a complex network. Such a muscular architecture is likely more suitable for stirring rather than pushing the embryos and gametes through the Fallopian tube. Anat Rec 267:235–241, 2002. © 2002 Wiley-Liss, Inc. Key words: salpinx; smooth muscle cells; mare; ovum transport; scanning electron microscopy In previous studies (Vizza et al., 1991, 1995; Muglia et al., 1991a,b, 1992, 1996a,b, 1997a,b) we demonstrated how direct observation of the myosalpinx structure (after removal of the interstitial connective tissue) under a scanning electron microscope (SEM) can help resolve the problem of contradictory data in the literature, which results from observation of bidimensional specimens. The most recent studies based on the direct observation of myosalpinx architecture (Muglia and Motta, 2001) further emphasized the need to clarify to what extent, if any, the musculature deriving from the mesosalpinx (namely, the extrinsic (salpinx) musculature (EXM)) is integrated in the salpinx musculature (intrinsic musculature) (INM)). This could help to ascertain whether the EXM has a role in the transport of gametes in addition to the functions it has in the pick-up of the oocyte from the ovary surface, and in the “tube-locking” phenomenon (Blandau, 1969, 1973). The literature available on EXM and INM of the mare is based on bidimensional observations under light microscopy (LM). Bignardi (1948) described in the tubo-uterine junction (TUJ) and the isthmus an INM constituted of isolated, loosely distributed, circular bundles that intermingle with longitudinal bundles in the innermost layer. These intermingled bundles form an inner architecture. © 2002 WILEY-LISS, INC. This inner architecture is lacking in the ampulla, which shows a unique circular orientation of bundles. Schilling (1962) suggested that the myosalpinx (INM) of Ungulates may be constituted by spiral fibers running deeper from the surface toward the base of mucous folds. The variable pitch of such spirals would account for the differences in the architecture of the myosalpinx between the tubal segments. Finally, Sisson (1973) described an outermost longitudinal layer (continuous with the muscle present in the mesosalpinx); an innermost intrinsic circular layer in the TUJ, isthmus, and ampulla; and a muscular sphincter in Grant sponsor: University of Messina; Grant number: PRA 2222/1998. *Correspondence to: Prof. Ugo Muglia, Department of Morphology, Biochemistry, Physiology and Animal Productions, Veterinary Faculty, University of Messina, Polo Universitario Annunziata, 98168 Messina, Italy. Fax: ⫹39-90-355246. E-mail: firstname.lastname@example.org Received 14 December 2001; Accepted 27 March 2002 DOI 10.1002/ar.10105 Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com). 236 GERMANÀ ET AL. the TUJ (INM). In consideration of these controversial data, as well as of the key role that the muscular architecture of a hollow tubular organ such as the Fallopian tube plays in the mechanism of its contraction, we investigated the three-dimensional (3D) architecture of the myosalpinx in the mare, with the aim of settling the controversy on this subject with definitive data. MATERIALS AND METHODS Sixteen pluriparous mares, aged 8 years, were used. The Fallopian tubes, collected immediately after slaughter, were dissected under a stereomicroscope and gently stretched and mounted by means of thin needles on a silicon plate in Krebs solution. This procedure was followed in order to observe the entire tube, respecting its topography. The tubes from 12 mares (six in estrous and six in anestrous) were processed for SEM. After dissection, the Krebs solution was replaced by 2.5% glutaraldehyde in 0.1 M phosphate buffer for 48 hr. The following segments were isolated from the stretched tubes, according to current anatomical and physiological classifications (Nilsson and Reinius, 1969): TUJ, isthmus, and ampulla. The segments were incubated in 6N NaOH at 60°C, according to Takahashi-Iwanaga and Fujita’s (1986) maceration technique, for 25 min. The digestion time was determined by trial and error. After chemical treatment, fragments were dehydrated in a graded series of alcohol, critical-point dried, coated with 20 nm of gold-palladium, and examined under a Cambridge Stereoscan 240 (SEM) at 20 kV. A number of samples were further microdissected by ultrasonication during dehydration (Low, 1989) for 3–5 min at 20 kHz, in order to reveal the deepest muscular bundles. The tubes from the remaining four mares (two in estrous and two in anestrous) were processed for light microscopy (LM). After they were dissected and stretched under the stereomicroscope, they were fixed in 10% formaldehyde, dehydrated in a graded series of alcohol, and embedded as a whole in historesin. From the resin blocks, 5-m transverse sections were taken and stained with hematoxylineosin (H&E). OBSERVATIONS The stretched tubes in the mare average 25 cm in length. Two muscular components can be distinguished within the myosalpinx: 1) a musculature running within the subperitoneal connective tissue (SCT) of the mesosalpinx (EXM), and 2) a musculature peculiar to the salpinx itself (INM)) (Fig. 1). In the following sections the myosalpinx architecture is described going from the TUJ to the ampulla, following the gradual decrease of its thickness. Comparative observations between specimens collected from estrous and anestrous mares did not show any difference in the described myosalpinx architecture in any segment of the tube (TUJ, isthmus, and ampulla). TUJ Extrinsic musculature SEM. The fibers are elongated, regularly outlined (in a relaxed stage), and joined in single, thick, loosely distributed, cylindric bundles, which follow a roughly longitudinal or oblique course. These bundles originate from the mesosalpinx, and reach and run along the surface of the underlying INM; they often bifurcate and anastomose along this course (Figs. 2 and 3). LM. In the outer musculature of the salpinx, the bundles observed under SEM appear, in transverse sections by LM, cut transversely or obliquely. These bundles are mixed together with the musculature present within blood vessel walls sectioned along different cutting planes (Fig. 6). Intrinsic musculature SEM. At different levels, bundles of EXM musculature of various length describe wide curves, changing their orientation and distributing transversely with respect to the major axis of the tube at different levels (Fig. 4). These bundles unravel at their extremities, merging into a circular compact coat (Fig. 5). The latter, therefore, appears mixed at different levels together with longitudinal and oblique bundles of EXM musculature. In a relaxed stage, the fibers of these bundles appear elongated and regularly outlined. The blood vessels that run within the myosalpinx are enveloped by a dense coat of irregularly outlined, roughly longitudinal muscle fibers (intrinsic vascular musculature) independent from the surrounding INM. This intrinsic vascular coat sometimes envelopes two parallel vessels. LM. Observed by LM, the wide, curved bundles mixed into the compact circular coat observed under SEM appear in transverse sections of the tube cut longitudinally or obliquely mixed at different levels inside a circular coat (Fig. 6). Furthermore, the intrinsic vascular muscle coats can be seen tangentially cut for a variable length within the INM. Isthmus Extrinsic musculature SEM. The EXM appears constituted (as in the TUJ) by elongated, quite regularly outlined (in a relaxed stage) muscle fibers joined in isolated, variously oriented bundles that are more densely distributed than in the TUJ. These cylindrical bundles lean against the periphery of the INM and anastomose (Fig. 7). The blood vessels that run within the myosalpinx show a musculature with features similar to those of the TUJ. LM. The myosalpinx shows the same basic structure as in the TUJ, although it is thinner and has less obliquely cut bundles (Fig. 9). Intrinsic musculature SEM. The elongated fibers form cylindrical bundles. As in the TUJ, these show an outer roughly longitudinal course and change to an inner direction. They follow a wide curve, distributing transversely or obliquely with respect to the major axis of the tube (Fig. 8), and unravel at their extremities, forming a roughly circular coat. The fiber bundles can often be seen leaving from the main bundles to form small plexuses. The blood vessels that run within the myosalpinx show an INM with features similar to that in the TUJ. LM. The myosalpinx appears less thick with respect to the TUJ. The bundles described by SEM appear constituted, in transverse sections, by segments of oblique bundles immersed in a coat of circular fibers. Fig. 1. General view of the intramural (i) and extramural (e) portions of the myosalpinx of the TUJ: uterine horn (u), peritoneum (p), and subperitoneal connective tissue (s). SEM, 20⫻. Fig. 2. Extramural portion of the TUJ. EXM: longitudinal and oblique muscle bundles (asterisks). SEM, 120⫻. Fig. 3. Extramural portion of the TUJ. EXM: bundles of SMC fibers bifurcate and anastomose repeatedly (asterisk), merging into the underlying INM (arrows). SEM, 310⫻. Fig. 4. Extramural portion of the TUJ. EXM bundles (arrow) of SMC fibers describe wide curves, change their orientation, and merge into the underlying musculature. SEM, 210⫻. Fig. 5. Extramural portion of the TUJ. INM: SMC fibers of the circular coat. SEM, 1430⫻. Fig. 6. Comparative histology of the Fallopian tube. Transverse section of the extramural portion of the TUJ: longitudinal and oblique muscle bundles (arrows), wide curves of muscle bundles (twin arrow), and circular muscular coat (asterisks). LM, H&E, 140⫻. 238 GERMANÀ ET AL. Fig. 7. Isthmus. Extrinsic musculature: oblique muscle fiber bundles (asterisks), peritoneum (p), and subperitoneal connective tissue (s). SEM, 380⫻. Fig. 8. Isthmus. Intrinsic musculature: SMC fiber bundles change their orientation from outer, roughly longitudinal to inner circular (double arrow). SEM, 90⫻. Fig. 9. Comparative histology of the tube. Transverse section of the isthmus. Extrinsic musculature: isolated, oblique muscle bundles (arrows). Intrinsic musculature: circular coat (double arrow) mixed with oblique bundles (asterisks). Subperitoneal connective tissue (SCT). LM, H&E, 340⫻. Fig. 10. Ampulla. Plexiform architecture of the intrinsic musculature. SEM, 530⫻. 3D ARCHITECTURE OF THE MYOSALPINX IN THE MARE 239 Fig. 11. Comparative histology of the tube. Transverse section of the ampulla. Loose SCT; intrinsic musculature: muscle fiber bundles cut along different planes; mucous epithelium (asterisk). LM, H&E, 160⫻. The diagram summarizes the 3D architecture of the myosalpinx as revealed by SEM after maceration. J, extramural segment of the TUJ; I, isthmus; A, ampulla. Extrinsic musculature (arrows); intrinsic musculature (i). Ampulla Extrinsic musculature SEM and LM. Only rare, isolated muscle fibers are observed. Intrinsic musculature SEM. The bundle and fiber shapes are similar to those observed in the isthmus. These loosely distributed bun- dles run across multiple planes and intersect, giving rise to a plexiform architecture (Fig. 10). The blood vessels are enveloped by the muscular coat, as previously described in the isthmus. LM. The musculature is considerably decreased in thickness compared to the previously described segments. The bundles of fibers observed under SEM as plexiform structures appear to be constituted by oblique segment fibers under LM (transversal section) (Fig. 11). 240 GERMANÀ ET AL. DISCUSSION The myosalpinx is generally constituted by an EXM originating from the broad ligament, and by a more conspicuous component that is peculiar to the myosalpinx (INM) (Muglia and Motta, 2001). In the mare, these two components are represented by a unique muscular structure that runs from the mesosalpinx to the base of the inner mucous folds (see diagram, Fig. 11). In this mammal, therefore, the terms EXM and INM have exclusively a topographic meaning. In this muscular complex our results show an EXM composed of single, thick, oblique and/or rather longitudinal bundles of fibers (described by Bignardi (1948) as plexiform, and by Sisson (1973) as longitudinal), which gradually lose their individuality as they reach the ampulla. Here the EXM consists of rare, isolated fibers. The INM consists of plexiform, loosely distributed bundles in both the isthmus and ampulla, which form an inner, compact, circular coat in the TUJ and isthmus. These bundles have been described as circular, or circular and longitudinal (Sisson, 1973), or (in Ungulates) as spirals (Schilling, 1962). However, the results of the present SEM study support our previous observations in the cow, sheep, and sow (Muglia et al., 1996b, 1997a,b), suggesting that the architecture of the IXM and EXM myosalpinx in the mare is mainly plexiform. The present findings do not invalidate the available data on the myosalpinx architecture in the mare; rather, they complete the data by providing a clear, topographical, 3D view of these features. In fact, the circular coat, the outer circular and inner longitudinal layers, and the spiral architecture reported by Sisson (1973), Bignardi (1948), and Schilling (1962), respectively, in the TUJ, isthmus, and ampulla correspond (according to our SEM results) to those oblique bundles of variable length and direction that run across multiple cellular planes and form a plexiform structure. Therefore, the disagreement in the literature about the structure of the mare myosalpinx may very likely arise from different interpretations of histological data. In fact, the observation of bidimensional sections by LM may be misleading when a 3D plexiform structure such as that of myosalpinx has to be described exactly. The muscle fiber bundles may appear obliquely, longitudinally, or unevenly circularly arranged depending on the plane of the sections, which only rarely are perfectly transverse. Moreover, the relative proportion of fibers and bundles following different spatial directions within a transverse section of a plexiform structure (such as the myosalpinx) may vary greatly, further affecting interpretation of the data. On the basis of our observations, and in relation to the morpho-physiological classification by Muglia and Motta (2001), the mare has a sphincter-like, type-b TUJ (plexiform and with a unique, complex EXM-INM) like that in the woman and cow, and a type-2 isthmus (plexiform and with a unique, complex EXM-INM) like that in the rabbit, ewe, and sow, as well as in the woman and cow. The sphincter-like TUJ is characteristic of species that have an intravaginal deposition (in the horse the penis does not intrude beyond the vagina (Day, 1942; Parker et al., 1975)) so that the uterus and the uterine horns are not directly subjected to the pressure of semen during mating. Therefore, a first selection of sperm is performed by the uterine cervix (Mann et al., 1956; Hunter, 1973; Polge, 1978). Thus, the TUJ, rather than functioning as a barrier against the flow of seminal plasma (as in the sow, which has a barrier-like TUJ) may have a more active role in modulating the seminal flow, as evidenced by its complex muscular architecture. In the type-2 isthmus the close fusion of the EXM with the INM is in accord with the existence of a unique mesosalpinx contractile system. Therefore, one may reasonably assume a direct control of the EXM over the INM in type-2 salpinxes (as in the rabbit, ewe, sow, cow, and woman). This supports the theory (Blandau, 1969, 1973) that the EXM has a primary role in the transport of gametes, in addition to its role in the “tube-locking” phenomenon. It is widely accepted that myosalpinx contractions propagate randomly, producing a backward–forward egg motion (Daniel et al., 1975a,b; Talo and Hodgson, 1978), and are transmitted, usually over short distances, from different pace-maker sites (Talo and Pulkkinen, 1982). These data were also confirmed by studies that recorded the random myoelectrical activity of the tube (Daniel et al., 1975a; Hodgson et al., 1977; Hodgson and Talo, 1978; Talo and Hodgson, 1978). Our observations show that the myosalpinx architecture of the mare (unlike that of hollow organs with geometrically arranged musculature (e.g., gut) in which the orthogonal disposition of the smooth muscle cells (SMC) is suitable to generate and coordinate peristaltic movements in an antagonistic manner) is similar to that of other hollow organs with plexiform musculature (e.g., the gall bladder) (see Uehara et al., 1990, for review). The contraction of such a plexiform SMC structure (Hodgson et al., 1977) may deform the tube wall, generating a stirring process within the tubal lumen. As a result of this stirring movement, the contact between the hormones and nutrients contained in the tubal lumen, on the one hand, and the gametes, zygotes, and embryos, on the other hand, is intensified, resulting in correct fertilization and early embryo development (Motta et al., 1994a,b, 1995, 1998, 1999). 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