The mucocutaneous junction and its associated nerves, immunoreactive cells and changes with age: a review with novel observations from human fetal and cadaveric specimens

Introduction

The mucocutaneous junction (MCJ) is a specific site located at body orifices such as the mouth, nose, eyelid, vulva, prepuce, and anus. Classically, the MCJ epithelium was known to show desquamation with a lack of both desmosomal contact and a lining of microplicae that anchor the polyanionic glycocalyx (e.g., ^[1] ). Later, Knop et al. ^[2] and Tektaş et al. ^[3] newly defined the MCJ as having both para-keratinized cells (“pk cells” containing, but not totally filled with, bulky bundles of keratin filaments) and discontinuous para-keratinized epithelial cells. Thus, the concept of the MCL is essentially supported by electron microscopy observations. Conversely, however, identification of the MCJ was often difficult using routine histology, especially in human cadaveric specimens, due to delayed fixation or long-term preservation.

Recently, in studies of nerves in the lip ^{[4, 5]}, we have found that routine histology of specimens from near-term human fetuses can clearly demonstrate the MCJ (Fig. 1). This prompted us to attempt a comparison of skin and epithelial morphology in near-term fetuses. Cytokeratin polypeptides (CKs) show organ-specific and epithelial type-specific expression (Table 1). The human skin epidermis strongly expresses CK1 and CK10 ^[6]. CK7 is a specific marker of the urogenital tract epithelium even in early fetal development ^{[7, 8]}, although in larger fetuses the urogenital sinus-derived structure is positive for CK14 and CK17 rather than CK7 ^[9]. CK5 and CK6 (usually abbreviated as “CK5/6” because of the specificity of commerciallyobtained antibodies) are used as markers of foregut-derived structures ^{[10, 11]}.

Therefore, CK immunohistochemistry is a commonly accepted approach for studies of the MCJ ^{[2, 3]}. However, on sections from human cadaveric specimens, CK immunohistochemistryoftenyieldspoorresults(inconsistent activity or loss) possibly due to delayed fixation or long-term preservation. On the basis of our previous studies using human cadaveric materials ^{[12, 13, 14]}, we considered that a combination of CK5/6, CK14 and CK19 would be most useful. Langerhans cells (a type of dendritic cell), CD8-positive suppressor T-lymphocytes and macrophages are distributed in and/or beneath the epidermis and mucosal epithelium. However, it seems that few data are available regarding any changes in their distribution and density at the MCJ. Omine et al. ^[15] performed a limited study of differences in the density of these cells between the skin and mucosa at the eyelid, mouth, prepuce and anus (Table 2).

Similarly, the distribution of free nerve terminals, especially those of pain nerves, and specific corpuscles and receptors has been described at limited sites, including the oral mucosa ^{[16, 17]}, nasal respiratory epithelium ^{[18, 19]}, glans penis ^{[20, 21]} and anus ^[22]. Conversely, few studies have demonstrated differences between the skin and the mucosa in the course (depth or lamination), density and terminal morphology of subcutaneous and submucosal nerves, except for the glans penis ^[23] and lower lip (^[4], Table 3). Overall, in contrast to cytokeratin expression, there is little information about possible morphological changes in nerves and immunoreactive cells at the MCJ. In the present study, on the basis of our previous studies as well as some new histological findings, we aimed to clarify histological changes at the MCJ in terms of 1) cytokeratin expression, 2) the distribution of Langerhans cells and other immunoreactive cells, 3) the course of nerves and terminals beneath the epithelium, and 4) transient morphologic changes in fetuses and suggested degenerative features associated with age.

Materials and Methods

This study was conducted in accordance with the provisions of the Declaration of Helsinki 1995 (as revised in 2013). Reviewed descriptions were based on our previous studies of the eyelids ^{[5, 15, 24, 25]}, the lip and mouth ^{[4, 15, 26]}, muscles at the nasal orifice ^{[27, 28, 29]}, the glans penis ^{[15, 23]}, the vulva ^[30] and the anus ^{[31, 32, 33, 34, 35]}. Because the MCJ was not the focus of these previous studies, however, we re-examined sections that had been prepared at the time and, at some sites, we conducted additional immunohistochemistry using unstained sections (for methods, see the final subsection of Materials and Methods).

Furthermore, based on observations of newly-prepared histological sections from eight adult cadavers and seven human fetuses (for details, see the subsections below), a number of novel descriptions were also included in the present study. Adult cadaveric specimens We dissected eight cadavers (three men and five women; aged 78–96 yr) that had been donated to Tokyo Dental College, Tokyo, Japan, for research and education on human anatomy with the approval of the Tokyo Dental College committee for research use (No. 922-2). These cadavers had been fixed by arterial injection of 10% neutral aqueous formalin solution and stored in 50% ethanol for more than 3 months. The external nose including the deep mucosa as well as the vulva including the distal urethra were removed from the cadavers and, using tissue blocks, routine procedures for paraffinembedded histology were performed.

Most sections were stained with hematoxylin-eosin (HE), azan, Masson trichrome or elastica Masson, while a smaller proportion was used for immunohistochemistry (see the final paragraph below). With elastica Masson staining (a variation of Masson-Goldner staining ^{[36, 37]}), elastic fibers are stained black, collagenous fibers appear green and striated muscles are bright red. All photographs were taken with a Nikon Eclipse 80. Human fetus specimens The seven late-stage fetuses were part of the collection kept at the Department of Anatomy, Akita University, Akita, Japan. They had been donated by the families concerned to the Department in 1975–1985 and preserved in 10% w/w neutral formalin solution for more than 30 years. Data for these specimens included the date of donation and the number of gestational weeks, but did not include the name of the family, obstetrician or hospital, or the reason for abortion.

The use of these specimens for research was approved by the Akita University Ethics Committee (No. 1428) and the sixth author (G.M.) is one of members of the research project. The research ethics committee of Tokyo Dental College also approved the use of the materials (No.932-2). The whole head of each fetus was obtained after removal of the brain and cutting below the hyoid bone as well as the pelvic region below the iliac crest, and after cutting at the midportion of thigh. Before routine procedures for paraffin embedding, the head and pelvic tissue masses were decalcified by incubating them at room temperature in Plank-Rychlo solution (AlCl2/6H₂O, 7.0 w/v%; HCl, 3.6; HCOOH, 4.6) for 3-7 days.

The sectional planes were sagittal (2 specimens), frontal (3) or horizontal (2). Most sections were stained with (HE), azan or Masson trichrome, while a minor part was used for immunohistochemistry (see below the final paragraph). All photographs were taken with a Nikon Eclipse 80. Immunohistochemistry of fetal and elderly adult specimens The primary antibodies used were 1) rabbit polyclonal antibody for S100A1 protein (Dako Z0311; Dako, Glostrup, Denmark), 2) mouse monoclonal antibody for vasoactive intestinal polypeptide (VIP) (sc25347; Santa Cruz; Santa Cruz, CA, USA), 3) mouse monoclonal antibody for cytokeratin-19 (sc-6278; Santa Cruz), 4) mouse monoclonal antibody for cytokeratin-14 (LL002; Novocastra, Newcastle upon Tyne, UK), 5) mouse monoclonal antibody for cytokeratin 5/6 (M7237, Dako), 6) mouse monoclonal antibody for CD1a (M357101-2, Dako), 7) mouse monoclonal antibody for CD8 (N1592, Dako) and, 8) mouse monoclonal antibody for chromogranin A (M0869, Dako).

9) mouse monoclonal antibody for alpha smooth muscle (M0851, Dako). For cytokeratin and CD-family antigens, antigen retrieval was performed with microwave treatment (500W, 15 min, pH 6). The secondary antibody (incubation for 30 min; dilution 1:1000; Histofine Simple Stain Max-PO, Nichirei, Tokyo) was labeled with horseradish peroxidase (HRP), and antigen-antibody reactions were detected using diaminobenzidine as the chromogen (incubation for 3-5 min; Histofine Simple Stain DAB, Nichirei, Tokyo). All samples were counterstained with hematoxylin. Negative controls consisted of samples without primary antibody.

Results

Data from references and our previous studies are summarized in four tables: cytokeratin expression (Table 1), immunoreactive cell distribution (Table 2), nerve course and terminal morphology beneath the skin-MCJ-mucosa (Table 3), and distinct changes with age in the surface histology and composite cells (Table 4). These tables overlap the text descriptions below somewhat, and sometimes differ from our novel findings reported here. To address these differences, the reader is directed to “See also the subsection entitled XX in the Results” in the table footnotes. When references are limited in number, especially for the nose and genitalia, appropriate newly-taken photos are provided (i.e., Figs. 1, 4, 5, 8 and 9).

Table 1. Expression of cytokeratin polypeptides (CK) in the skin, mucosaand MCJ. Skin MCJ mucosa Lip1) Nose2) Eyelid3) Urethra4) Anus5) CK1,10-positive CK13-positive CK10-positive CK10-positve CK5,10,13-positive CK14-positive CK13-positive CK10-positive, CK19-negative CK7-positive, CK10-negative CK19-positive CK14,19-positive CK13-negative CK13,19-positive CK7-positive CK8,18,19-positve References: 1) Barret et al. ^[60](see also a subsection entitled “Lips and mouth” in the Results); 2) Kasper et al. ^[61], Schwerer et al. ^[62]. See also a subsection entitled “Nasal orifice” in the Results. 3) Tektaş et al. ^[3]. 4) Herrera-Imbroda et al. ^[7], Shen et al. ^[8]. 5) Levy et al. ^[63], Williams et al.^[64] Research Article Table 2. Langerhans’ cell and other immunoreactive cells in the skin, mucosa and MCJ Skin MCJ mucosa Lip1) Nose2) Eyelid3) Vulva, prepuce4) Anus5) Langerhans+ CD8+ >or< CD4?

DC+ Langerhans+ Langerhans++ Langerhans+ Langerhans++ CD8+ > CD4+ no distinct change MCJ-M, gradual change Langerhans absent gradual change gradual change MCJ-M, drastic change Langerhans+ Langerhans++ CD8+ > CD4 DC+ Langerhans+or CD4+ > CD8+ DC+ Langerhans+ CD8+ > CD4+ CD8+ in epithelium; CD4+ in lamina propria Langerhans- Langerhans CD8+ > CD4+ DC+ DC, non-Langerhans type dendritic cells such as CD209 (DC-SIGN)-positive cells. References: 1) Mechtersheimer et al. ^[65], Cruchley et al. ^[66], Omine et al. ^[15], Reinartz et al. ^[67]. 2) Yoshimi et al. ^[68], Graeme-Cook et al. ^[69], Kawahara et al. ^[59], Takasaki ^[70], Jahnsen et al. ^[40], Morinaka et al. ^[71], Jahnsen et al. ^[41], Karosi et al., 2013.

See also a subsection entitled “Nasal orifice” in the Results. 3) Hingorani et al. ^[73], Omine et al. ^[15]. 4) Morelli et al. ^[74], Omine et al. ^[15], Qin et al. ^[75]. 5) Mechtersheimer et al. ^[65], Gervaz et al. ^[76], Omine et al. ^[15]. Table 3. Nerve course and terminal morphology in the skin, mucosa and MCJ Skin mucosa MCJ Lip1) corpuscle-like terminal along the dermal papilla free end of twigs parallel to surface Merkelneurite complex absent varicose-like structure, mechanoreceptors drastic change Nose2) free end substance P+ nerve free end substance P+ nerve other peptidergic nerves distinct change to M free end? Eyelids3) corpuscle-like terminal, free nerves parallel to epithelium free end distinct change to M corpuscle-like terminal Vulva, prepuce4) free end dominant Meissner’s corpuscles free end distinct change in density Anus5) free end Myenteric plexus abundant ganglia gradual but distinct change free end, a few ganglia Pacinian corpuscle References: 1) Ramieri et al.

^[16], Hilliges et al. ^[77], Watanabe ^[17], Cho et al. ^[5]. 2) Lundblad et al. ^[18], Zhao et al. ^[78], Hauser-Kronberger et al. ^[19]. See also a subsection entitled “Eyelids” in the Results. 3) Cho et al. ^[4]. Research Article 4) Halata et al. ^[20], Hinata et al. ^[79], Kurokawa et al. ^[30], Jang et al. ^[23], García-Mesa et al. ^[21]. 5) Li et al. ^[22], Hieda et al. ^[33], Ishiyama et al. ^[34], kinugasa et al. ^[35]. Table 4. Distinct changes with age in the surface histology and composite cells Skin mucosa MCJ Lip1) lost cornification6) difference in nerve course8) Nose2) lost cornification elongation Eyelids3) lost stratification after birth7) some nerves disappear9) Vulva, prepuce4) lost cornification pseudostratified to squamous Anus5) lost cornification elongation with fetus anal sinuses involved immunoreactive cell infiltration, venous plexus proliferation References: 1) Cho et al.

^[5]. 2) See also a subsection entitled “Nasal orifice” in the Results. 3) Cho et al. ^[4]. 4) See also a subsection entitled “Vulva and prepuce” in the Results. 5) See also a subsection entitled “Anal canal” in the Results. 6) Lost cornification: the cornification corresponds to a highly increased stratification with hypertrophied keratinized cells (Kim et al. ^[14]). A transient closure of the lumen is evident in the external auditory meatus and nasal orifice (Fig. 3i) and also seen at the anus and male urethral orifice (Fig. 9C). 7) In fetuses, the conjunctival epithelium is stratified cuboidal, not simple cuboidal. 8) A sudden change in nerve course and terminal at the MCJ of lips becomes evident possibly after birth (Cho et al.

^[4]). 9) Subepithelial nerve twigs in parallel to the MCJ epithelium of eyelids were seen in fetuses (Cho et al. ^[5]), but they were not reported in adults Lips and mouth The lips showed the typical changes in MCJ morphology immediately outside or anterior to the approximation surface (free surfaces attached to each other between the upper and lower lips). Thus, the approximation surface corresponded to the mucosa. The change was marked at near-term because, in the mucosa with hypertrophic surface cells, the stratification was 6-7 times taller or thicker than that of the skin (Figs. 1j and 2h). According to Cho et al. ^[4] and our re-examination for the present study, the lip skin epidermis in elderly individuals expressed cytokeratin-14 (CK14) at any depth, while CK14 positivity was limited to the most superficial lamina of the stratified epithelium of the mucosa and MCJ (Fig.

2cd). Thus, the lip skin exhibited sudden loss of strong CK14 positivity at the MCJ. CK5/6 and CK19 were negative in three parts of the lip in elderly individuals. However, these results differ from those in the references (Table 1). Langerhans cells were richly distributed in the stratum spinosum, while CD8-positive T lymphocytes were dominant in the lamina propria beneath the mucosal epithelium (Fig. 3). In the elderly, the lip mucosa lacked a tall stratified layer, often resembling simple squamous epithelium (Fig. 2a). Near the free margin of the lower lip, a thick branch of the mental nerve turned superficially (anteriorly) to pass through the upper end of the orbicularis muscle layer to supply the lip skin, whereas another thick branch took a reverse J-shaped course along the stratified squamous epithelium of the free margin and reached the MCJ.

In the subcutaneous tissue, nerve twigs were concentrated along and around veins and dermal papillae. At the MCJ, terminal nerve twigs running in parallel to the mucosal surface were replaced by capsule-like nerve terminals attached to or surrounding internal protrusions of the skin stratum spinosum between the dermal papillae. This change in nerve terminals was much more evident in elderly individuals than in fetuses (Fig. 2e-g). Overall, in the fetal lips, the mucosa was characterized by 1) very thick stratification of the cornified layer and 2) a short and abrupt change at the MCJ where thick cornification was lacking (Table 5). As nerve twigs are likely to be underdeveloped even in late-stage fetuses, a drastic change in nerve terminals at the MCJ would likely occur after birth or when suckling is initiated.

Table 5. Site-specific morphologies of the skin-MCJ-mucosa in fetuses Skin MCJ area mucosa Lip1) hairs short & sudden change very thick stratification Nose hairs; striated muscle insertion to the dermis long and wide, but drastic at MCJ- Mucosa, extending with age columnar with cilia Eyelids2) eyelash; Skin-MCJ, a small difference in stratification MG duct opening cuboidal with vacuole Vulva, prepuce3) thick stratification, no hairs, no dermal papillae long and wide similar to stratified squamous cavernous sinus & smooth muscle pseudostratified pale-colored tall cells Anus4) abundant small dermal papillae no hairs facing the lumen long and wide, but drastic at MCJ- Mucosa dilated veins columnar with goblet cells 1)In the lips, lost thick stratification of the oral mucosa seemed to occur after birth: but this change seemed not to induce an internal extension of the skin.

2) Meibomian gland (MG) epithelium is similar to and continues to the MCJ. 3) In the vulva and prepuce, a long and wide MCJ (possibly underdeveloped pseudostratified but similar to the stratified squamous) seems to be maintained after birth. 4) The anal transitional zone epithelium initially develops as the fetal anal sinus that widen a circumference of the anal canal prenatally. Thus, the anal “MCJ” in adults does not develop as a junction between the skin and mucosa but as an independent epithelium derived from a secondarily-added structure. Nasal orifice The nasal mucosa is a pseudostratified columnar epithelium with cilia (Figs. 1k, 4ch and 5ek), although the cilia were usually damaged due to long lag-time after death in the present materials.

The MCJ was short and composed of a thin stratified squamous epithelium at near-term and with few goblet cells in elderly individuals (Figs. 1g, 4d and 5d). The skin was very long and wide in the elderly and often reached a depth of more than 30 mm within the orifice (e.g., almost 20 mm in Fig. 5). The MCJ was usually lower or more superficial in the medial (septal) side than the lateral (alar) side. The nasal skin was characterized by striated muscle fibers contained; near-term specimens clearly demonstrated nasalis muscle fibers passing between the glands or hairs and inserting deeply to the dermis (Fig. 4). In fetuses at 10-16 weeks, the nasal orifice was transiently closed by cornification or hypertrophic keratinized cells ^[14] (Fig.

3i). The nasal MCJ, as thick as the nasal skin epidermis, had no or few internal protrusions of the stratum spinosum (or the corresponding dermal papillae) and lacked hairs and nasalis muscle. Instead, a vein-rich, loose tissue was present beneath the MCJ. Cytokeratin-13ispositiveintheskinaswellastheMVJbutnegativeinthenasalmucosa(Table1).Weobservedthatcytokeratin-14 reactivity was strong on the basal side of the epidermis, weak at the MCJ, and very weak in the mucosal epithelium (Fig. 5c-e). Acinus cells beneath the mucosa were positive for cytokeratin-14. Nerves were few beneath the MCJ and mucosa (Fig. 4h). However, in the subcutaneous tissue, abundant nerves took wavy courses while maintaining an almost constant depth of 1 mm beneath the epidermis (Fig. 4g). Thus, the skin nerves crossed striated muscle fibers at almost right angles.

Langerhans cells were rich in the skin epidermis, especially in and along hair follicles, but absent at the MCJ and in the mucosa (Fig. 5f-h). In the mucosa, it had been reported that CD4-positive T lymphocytes were more dominant than CD8-positive cells. However, we found only a few CD4-positive T lymphocytes at all sites near the nasal orifice. Mononuclear cells including CD8- positive T lymphocytes had infiltrated beneath the epidermis and MCJ, but were absent in the mucosa (Fig. 5i-k). Notably, CD8- positive cells were seen in both the epidermis and columnar epithelium (Fig. 5i and k). These lymphatic tissues appeared not to correspond to the nasal-associated lymphoid tissue (NALT) that has been defined in animals.

To our knowledge, studies of human NALT have not focused on areas at and near the MCJ but rather deep areas near the pharyngeal and tubal tonsils ^{[38, 39]}. The human NALT contains not only subtypes of macrophage and dendritic cells but also M cells ^{[40, 41]}. Overall, at the nasal orifice, the skin was characterized by striated muscle fibers inserting to the dermis. CD8-positive cells were present in both the epidermis and columnar epithelium. After birth, the MCJ is most likely to expand deeply (posteriorly and superiorly). Therefore, a deep and abrupt change between the MCJ and ciliated mucosa was often evident in aged individuals (Table 5). Eyelids The morphology of the eyelid MCJ has been described in detail by European groups (see the Introduction).

In short, the epithelium has been characterized by “para”- keratinization and discontinuity, and subsequently the MCJ was reported to correspond to the posterior or internal 1/3 of the approximation surfaces between the upper and lower lids. Thick ducts of meibomian glands opened to the MCJ and the duct epithelium appeared to supply epithelial cells to the MCJ. Riolan’s muscle (striated muscle; the inferoposterior end of the orbicularis oculi muscle) was located close to the MCJ epithelium. The duct was always dilated at near-term (Fig. 6a) because the opening was obliterated, possibly due to high cell proliferation rate of the MCJ. The palpebral skin and MCJ were cytokeratin10 (CK10)- positive, while the conjunctival mucosa was cytokeratin19- positive (Table 1).

In near-term fetuses, the approximation surfaces of the eyelids contained abundant corpuscle-like nerveterminalsatthebaseoftheepithelium.Simultaneously, the lamina propria contained nerve twigs running in parallel to the epithelium (Fig. 7d). However, the parallel nerves are likely to be restricted beneath the approximation surface ^[5] (Fig.7c). LangerhanscellswererichintheskinandMCJandfew in the mucosa. CD8-positive T lymphocytes were dominant beneath the epithelium at any site and depth (Table 2; Fig. 7ef). In elderly individuals, the entire approximation surface appeared to carry thick stratified squamous epithelium, but contained surprisingly few nerves. The surface morphologies in Fig 1chl (252 mm CRL) are quite different from those in Fig. 6 (270 mm CRL). The former (252 mm) upper lid, possibly poorly differentiated, carries a thin epidermis of palpebral skin, thick stratification at the approximation surface, and the cuboidal epithelium of the conjunctiva.

The latter (270 mm) is well differentiated and shows thick epidermal stratification (Fig. 6b), thin stratification of the approximation surface, and pseudostratified cuboidal epithelium of the conjunctiva (Fig. 6fk). The specific cuboidal epithelium contains vacuoles in the surface cells of the lower palpebral conjunctiva. The cells with vacuoles were considered likely to develop to goblet cells ^{[42, 43]}. In these large fetuses, it was rather easy to discriminate the “approximation surface epithelium” (not the MCJ) from either the skin or the mucosa. The typical MCJ as defined by European groups (see above) might be established after birth. Overall, in the fetal eyelids, any difference between the MCJ and thin palpebral skin was limited: 1) eyelashes and slightly thicker stratification in the skin and 2) meibomian duct epithelium continuous with the MCJ with obliterated ductal openings.

Similarly, a gradual change between the MCJ and conjunctiva was characterized by candidate goblet cells and intercellular slits in the cuboidal mucosal epithelium (Table 5). Thick stratification is not evident in the eyelids, especially at the approximation surface. Vulva and prepuce In 10-15 weeks at midterm, the epidermis showed a marked increase of stratification thickness at the deep skin groove of the vulva (Fig. 8b) and the skin surrounding the space between the prepuce and the glans penis (Fig. 9bc). The latter space around the glans was filled and obliterated with cornification (Fig. 9c) and epithelial pearl-like structures were present (Fig. 9b). Epithelial pearls are a well-known feature of the oral mucosa, especially in the palatal aspect ^[27].

The MCJ epithelium at the urethral orifice changes abruptly to a thin stratified squamous epithelium (Figs. 1i and 8egh) and continued to the pseudostratified columnar epithelium of the distal urethra (Figs. 1m, 8i, 9d and 10d). The squamous MCJ of the vulva was also connected to the highly stratified epithelium of the vagina (Fig. 8d). In adults, the vulva and glans penis are covered by a specifically thin epidermis without a lining of dermal papillae. The subcutaneous tissue contained a complex of cavernous venous sinuses (Figs. 8gh and 10a). Beneath the skin, nerve density in the vulva was much lower than in the prepuce (Fig. 8j vs. Fig. 10c), and corpusclelike nerve terminals were considered to be likely absent in females (Table 3).

The skin of both the vulva and prepuce was CK10-positive, while the MCJ and urethral mucosa were CK7- positive even in early fetuses. Langerhans’ cells were seen in the skin and MCJ, and CD8-positive T lymphocytes were richly present at any site and depth (Fig. 10ef). According to our interpretation of ^[44], the genital MCJ was considered likely to contain immunoreactive cells similar to those in the nasal MCJ in terms of density and subtype. The tunica albuginea enclosed the corpus cavernosus penis even in fetuses (Fig. 9a), but we found no evidence for blending of the tunica with the dermal papilla at sites distant from the glans as reported by Halata and Munger ^[20].

Overall, in both fetal and cadaveric specimens, the prepuce and vulva were characterized by 1) a lack of hairs and dermal papillae, 2) a long and wide MCJ with a specific surface, and 3) a cavernous sinus of veins and smooth muscles distributed beneath the skin and MCJ. The specific surface was covered by apparently underdeveloped pseudostratified columnar epithelium, although there was some resemblance to stratified squamous epithelium. As was evident in the urethra of elderly individuals, the MCJ in young adults might possess true pseudostratified epithelium comprising pale-colored tall cells (Table 5). Anal canal The anal skin had tall stratified layer at midterm (Figs. 1e and 11ef), but this was lost at near-term.

Conversely, the columnar epithelium became tall until near-term. From the rectal side to the skin side, the anal canal epithelium changed from columnar epithelium with abundant goblet cells (Fig. 11b), via cuboidal epithelium with few goblet cells (anal transitional zone=MCJ; Figs. 1n and 11c), to stratified squamous epithelium (anal skin; Figs. 1e and 11d). Being independent of both the squamous skin and columnar mucosa, the putative MCJ initially developed as an epithelium of the anal sinuses that considerably increased the circumference of the anal canal at midterm ^[32](Fig. 11f). The MCJ expresses SOX2 but the anal skin and mucosa do not ^[45]. The anal canal possessed a muscularis mucosae or anal submucosal smooth muscle (Figs.

11a and 12ad) between the mucosal epithelium and circular muscle layer (the latter representing the internal anal sphincter), but the submucosal muscle showed great individual variations in thickness and extent ^[31]. Beneath the MCJ, the submucosal muscle fibers disperse and disappear (Fig. 12e). The myenteric or intermuscular autonomic nerve plexus disappeared slightly orad to the MCJ (Fig. 11e). Thus, the skin side of the mucosa did not carry the nerve plexus, but ganglion cell clusters were sparsely distributed in the intersphincteric zone beneath the MCJ. Notably, submucosal nerve twigs are few or even absent beneath the MCJ in contrast to abundant nerves beneath the skin and mucosa (Fig. 12b). Conversely, chromogranin-positive neuroendocrine cells are few in the mucosa (Fig.

12f), absent in the skin (Fig. 12c) and rich at the basal layer of the MCJ (Fig. 12g). Hörsch et al. ^{[46, 47]} demonstrated chromogranin-positive elongated neuroendocrine cells distributing densely at the anal MCJ. Some of neuroendocrine tumors from the anal canal also express chromogranin A ^{[48, 49]}. In elderly individuals, the MCJ was characterized by infiltration of lymphocytes and macrophages and, beneath it, dilated hemorrhoidal veins were evident (Fig. 11ac). Langerhans cells were more numerous than CD8-positive lymphocytes in the anal skin and MCJ (two inserts in Fig. 11a). Overall, in the fetal anal canal, the skin was characterized by 1) a lack of hairs facing the lumen and 2) abundant small dermal papillae.

The MCJ is derived from the fetal anal sinus epithelium, being analogous to the eyelid MCJ derived from the meibomian duct. Anal glands are open to the anal MCJ ^{[45, 50]}, but the ducts are underdeveloped in fetuses. The long and wide MCJ observed in elderly individuals had accompanying well-developed venous plexuses and mononuclear cell infiltration. The anal MCJ is known to be a site where composite lymphoid tissues undergo a suppressive reaction under conditions of virus infection ^{[50, 51]}. Finally, intestinal nerve plexuses never extended to the MCJ (Table 5). FIGURES Figure 1. Mucocutaneous junction in near-term fetuses. The middle column of panels shows the mucocutaneous junction (MCJ). Panels a, f and j (Masson trichrome), lower lip of a 310-mm fetus; panels b, g and k (Masson trichrome), external nasal orifice of a 334-mm fetus; panels c, h and l (HE), upper eyelid and palpebral conjunctiva of a 252-mm fetus; panels d, i and m (HE), vulva and female urethra of a 214-mm fetus; panels e and n (HE), anal canal of a 265-mm fetus.

In the lower lip (panels a, f and j), note very tall stratified squamous epithelium with protrusions of the lamina propria mucosae (stars in panel j). Panel k displays ciliated pseudostratified columnar epithelium (respiratory epithelium) of the nose. In the upper eyelid, the MCJ morphology (panel h) is seen at the approximation surface in near-term fetuses. The distal female urethra (panel m) carries pseudostratified columnar-like epithelium at near-term: it is also similar to transitional epithelium in the male urethra. Tall stratification of the anal skin (panel e) is lost and goblet cells (arrows) appear in the MCJ of the anus (panel e) and, then, the latter changes into the columnar epithelium (panel n).

Asterisks in panel n indicate an artifact separation of the mucosa. Lower-magnification views are shown in Fig. 2h (lip), Fig. 4e (nose), Fig. 6a (eyelid), Fig. 8c (vulva) and Figs. 11ef and 12ac (anus). Panels a, f and j or panels b-d, g-i ,k-m and e and n were prepared at the same magnification (scale bars: 0.1 mm in panels a, b, e, f, j, n; 0.5 mm in panel e; 5 mm in panel n). Figure 2. Nerve distribution in the lower lip of a 75-year-old man: immunohistochemistry for S100 protein and cytokeratin 14 at the MCJ. Sagittal sections. HE staining (panel a); immunohistochemistry for S100 protein (panels b and e-g); immunohistochemistry for cytokeratin-14 Research Article (panels c and d).

Higher-magnification views of the squares in panel b (or panel c) are shown in panels e-g (or panel d). Panels a-c show adjacent sections. Thick nerve branches run anteriorly through the orbicularis oris muscle layer (OORM) toward the skin side of the lower lip (panel b). The stratum spinosum of the epidermis is strongly positive for cytokeratin 14 (CK14; panels c and d), in contrast to the mucosa. At the MCJ (panel f), the epidermis appears abruptly. The mucosa contains thin nerve twigs (arrows in panel e), while the stratum spinosum contains corpuscle-like nerve terminals at the base facing the dermal papillae (arrowheads in panels f and g). Panel h shows the near-term lip: the MCJ is clear because of the very thick stratified epithelium of the mucosa (see also Fig.

1j). Scale bars, 1 mm in panels a-c and h; 0.1 mm in panels d-g. Figure 3. Immunoreactive cells in the oral mucosa and lower lip skin from an 86-year-old woman. Panels a-d display the lower lip skin, while panels e-h show the oral mucosa near the lower lip. Panels c and d (or panels g and h) are highermagnification views of the squares in panels a and b (or panels e and f), respectively. The stratum spinosum of the lip skin contains abundant CD1-positive Langerhans cells (panel c) and a few T-lymphocytes (panel d). The oral stratified epithelium contains abundant Langerhans cells (arrows in panel g), while T-lymphocytes are restricted to the submucosal side of the mucosa (panel h).

Panel i shows a sagittal section (elastica Masson staining) of the nasal orifice of a 150-mm fetus and a star indicates cornification closing the orifice. AC, alar cartilage; MX, maxilla. Panels a, b, e and f or panels c, d, g and h were prepared at the same magnification (scale bars: 1 mm in panels a and i; 0.1 mm in panel c). Figure 4. MCJ at the external nasal orifice of late-stage fetuses. HE staining (panels a-f) and S100 immunohistochemistry (panel g and h). Panels a-d, a single frontal section from a 215-mm fetus; panels e-h, from a 334-mm fetus. Panels b-d (or panels f and h) are higher-magnification views of the squares in panel a (or panel e).

Panel g, a section near panel a, shows the site indicated by a circle in a. Panels g and h show immunohistochemistry for S100 protein. The skin (panels b and f) contains abundant glands, hairs and striated muscle fibers of the nasalis muscle (NM). The mucocutaneous junction (MCJ) is covered by stratified squamous epithelium (panel d) and, beneath it, loose fibrous tissue is associated. The nasal mucosa comprises stratified columnar epithelium (panel c) or respiratory epithelium (columnar epithelium with cilia; panel h). Note the linear arrangement of nerves (dotted line in panel g) in the nasal subcutaneous tissue. The nasal skin and epithelium (specimen same as that in panel e, 334-mm fetus) are also shown in Fig.

1b, g and k. Scale bars, 1 mm in panels a, e and g; 0.1 mm in panels b-d, f and h. Figure 5. Reactivities for cytokeratin, CD1a and CD8 in the skin, MCJ and mucosa near the external nasal orifice of adult cadavers. A 77-year-old woman. Panel a, elastica Masson staining; Panels b-e, immunohistochemistry for cytokeratin 14 (CK14); panels f-h, immunohistochemistry for CD1a showing Langerhans cells; panels i-k, immuno-histochemistry for CD8 showing suppressor T lymphocytes). Panels a and b display adjacent sections and their left-hand margins corresponds a site almost 20 mm deep or further within the external orifice of the nose. The hairs and associated glands disappear at a site 20-21 mm from the orifice entrance, while the columnar epithelium with goblet cells appears at a site 35-36 mm from the orifice entrance.

CK14 reactivity is strong in the basal part of the epidermis and weak or very weak in the MCJ and mucosa (panels c-e). Acinus cells beneath the mucosa express CK14 (panel e). Langerhans cells are sparsely distributed in the epidermis (panel f) and densely distributed in the hair follicles (panel g). The MCJ and mucosa do not contain Langerhans cells. Mononuclear cell infiltration (surrounded by dotted line in panels I and j) including CD8-positive T lymphocytes is evident beneath the epidermis and MCJ, but absent beneath the mucosa. Goblet cells are seen in the center of panel j. The epidermis and columnar epithelium contain CD8-positive cells (arrows in panels i and k).

Asterisks in panels b and d indicate tissue damage during the histological procedure. Scale bars: 1 mm in panels a and b; 0.1 mm in panels c-k. Figure 6. Identification of the skin-MCJ-mucosa in the upper and lower eyelids of a late-stage fetus (CRL 260 mm). HE staining. Panel a displays the topographical anatomy: 10 circles are shown in panels b-k at higher magnification. In the upper lid, thick stratification of the epidermis (black stars in panel b) changes to a thin layer (open star) at a site indicated by the arrowhead (candidate skin-MCJ junction). The thin stratification of the epithelium continues to panels d and e (open stars). In the epithelium panel e, intercellular spaces (arrows) appear superior to a site indicated by the arrowhead (candidate MCJ-mucosa junction).

Round nuclei (arrows in panel f) are arranged in line along the epithelial surface. In the lower lid, thick stratification (black stars) of the epidermis changes to a thin layer (open star; MCJ) at a site indicated by the arrowhead in panel g, and at a site indicated by the arrowhead in panel J, the MCJ connects to the stratified mucosa containing candidate goblet cells with vacuoles (arrows in panels j and k). Panels b-k were prepared at the same magnification. OOM, orbicularis oculi muscle. Scale bars: 1 mm in panel a; 0.1 mm in panel b. Figure 7. Nerve distribution at the MCJ in the upper eyelid of a near-term fetus and immunoreactive cells in an elderly individual: immunohistochemistry for S100 protein, CD1a and CD8 Panels a-d (immunohistochemistry for S100 protein) display a single sagittal section of the upper eyelid from a 252-mm fetus.

Panels e and f show immunohistochemistry for CD1a and CD8 at the MCJ of the upper lid in a specimen from an 86-year-old woman (same as shown in Fig. 3). Panels b-d (same magnification) are higher-magnification views of the squares in panel a. The nerve density is higher on the conjunctival side (panel c) than on the skin side (panel b). At the MCJ (panel d), a thin nerve runs along the anteroposterior axis (arrowheads), while corpuscle-like nerve terminals are scattered in the surface epithelium (arrows). Asterisks indicate tissue damage during the histological procedure. The MCJ and conjunctival mucosa contain abundant CD1a-positive Langerhans cells (panel e), but T-lymphocytes are restricted to the subcutaneous or submucosal tissue (panel f).

OOM, orbicularis oculi muscle; MG, meibomian glands. Panels b-d or panels e and f were prepared at the same magnification (scale bars: 1 mm in panel a, 0.1 mm in panels b and e). Figure 8. MCJ in the vulva: changes with age. HE staining (panels a-e), elastica Masson staining (panels f-i) and S100 immunohistochemistry (panel j). Panels a and b, a 155-mm fetus; panels c-e, a 215-mm fetus; panels f-j, a specimen from a 72-year-old woman. Panels b and e are higher-magnification views of the square in panel a, respectively, and they contain inserts showing the urethral mucosa 1 mm distal to the orifice. Panel d, a plane 0.2 mm posterior to panel c, shows highly stratified squamous epithelium of the vagina.

Panels g, h and j, corresponding to the circle in panel f, exhibit the urethral mucosa containing abundant veins (panel g) and smooth muscles (panel h) but few nerves (panel j). Panel i shows the distal urethral mucosa 5 mm distal to the orifice. Thick stratification (open stars in panel b) is evident at the approximation surfaces of the perineal epidermis in the early stage. A non-squamous epithelium with thick stratification (panels e, g and h) appears to correspond to the MCJ at the vulva. Scale bars: 1 mm in panel a, c, d and f; 0.1 mm in panels b, e, g, h, i and the insert in panel a.

Figure 9. Cornification filling the space between the prepuce and glans penis in a late-stage fetus. HE staining. Panels a-c display a 195-mm fetus (sagittal section), while panel d shows a 232-mm fetus (transverse section). Panels b and c, higher- magnification views of the squares in panel a, showing thick cornification (high stratification) closing the space between the prepuce and glans penis. Epithelial pearl-like structures are evident in the cornification (arrows in panel b). At this stage, the urethral mucosa is pseudocolumnar and the lamina propria contains abundant veins (panel d). CCP, corpus cavernosum penis; CS, cavernous sinus. Panels b-d were prepared at the same magnification. Scale bars: 1 mm in panel a; 0.1 mm in panel b.

Figure 10. Skin of the prepuce and glans penis of a 75-year-old man. Panel a, Masson trichrome; panels b-d, S100 protein immunohistochemistry; panel e, CD8 immunohistochemistry; panel f, CD1a immunohistochemistry. Panels a and b are adjacent sections. Arrow at the urethral orifice in panel A indicates an artifact (tissue fragment). Panels c and d are higher-magnification views of the circles in panel b, while panels e and f correspond to the circle at the lower angle of the prepuce in panel a. Abundant nerve twigs take highly tortuous courses beneath the penile skin (panel c) in contrast to only a few nerves along the urethra (UR; panel d). The penile skin contains many more CD8-positive lymphocytes than Langerhans cells (panels d and e).

CS, cavernous sinus of the veins beneath the skin and mucosa. Panels c and d or panels e and f were prepared at the same magnification. Scale bars: 5 mm in panels a and b; 0.1 mm in panels c and e. Figure 11. MCJ at the anal canal of an elderly individual and a late-stage fetus. Panels a-d, longitudinal sections of the lateral anal wall of a 68-year-old man; panel e, frontal section of a 160-mm fetus. Panels b-d (immunohistochemistry for S100 protein) show higher-magnification views of the squares in panel a. In the elderly individual (panel a), a long MCJ (usually called the “anal transitional zone”) extends between the columnar epithelium and skin epidermis.

The epithelium contains abundant mononuclear cells (panel d). Panel e (immunohistochemistry for vasoactive intestinal polypeptide (VIP)) exhibits a myenteric nerve plexus extending beneath the mucosa. A non-specific reaction is seen in the two smooth muscle layers. Panel f displays the developing anal sinus (origin of the future MCJ epithelium) in a 140-mm fetus. Two inserts of panel a show that Langerhans cells predominate over CD8- positive lymphocytes in the skin. EAS, external anal sphincter (striated muscle); IAS, internal anal sphincter (smooth muscle); SMM, submucosal longitudinal smooth muscle (muscularis mucosae). Panels b-d were prepared at the same magnification (scale bars: 1 mm in panels a and e and an insert, 0.1 mm in panels b-d).

Figure 12. Anal submucosal muscle (muscularis mucosae) and chromogranin-positive cells at the MCJ of the anal canal in a 195-mm fetus. Panels a, d and e, immunohistochemistry for smooth muscle actin (SMA); panel b, immunohistochemistry for S100 protein (S100); panels c, f and g, immunohistochemistry for chromogranin A. Panels d and e (or Panels f and g) are higher magnification views of squares in panel a (or panel c). The muscularis mucosae (stars) disperses to provide a thick but loose layer at MCJ (panel e). Panel B displays few or absent nerve beneath the MCJ in contrast to abundant nerves at the submucosal and subcutaneous area (arrows). Chromogranin A-positive neuroendocrine cells are few in the mucosa (panel f) but rich at the basal layer of the MCJ (panel g).

scale bars: 1 mm in panels a-c, 0.1 mm in panels d-g.

Discussion

We re-examined the morphology of the MCJ in humans to clarify similarities and differences among five sites (Tables 1-4) and, especially in fetuses, we considered what features might be site-specific (Table 5). In the eyelids, nasal orifice, urethral orifice and anus, the MCJ is a transitional area in which the skin epidermis connects to a non-squamous epithelium. Therein, the developing MCJ epithelium in fetuses was more or less similar to a stratified squamous epithelium rather than a columnar, cuboidal or pseudostratified columnar epithelium. Pseudostratified epithelium is known to show an intermediate morphology with active proliferation, differentiation and nuclear movement, and in embryos it covers various sites including the neural groove and canal ^[52].

The fetal MCJ we observed might correspond to a stage of development successive to the embryonic pseudostratified epithelium. Although they are usually found intercalated within a simple columnar epithelium, goblet cells are interspersed within a stratified MCJ at the eyelid ^{[2, 43]} as well as the anal canal ^[45]. The present figures demonstrated not only their candidates (Figs. 1en and 6jk) but goblet cells at the stratified MCJ of the elderly nose (Fig. 5j). Notably, at the eye and anus, goblet cells are most likely to secret a very large and heavy O-glycosylated mucin MUC5AC ^{[43, 45]}. The nasal and male urethral orifices were transiently obliterated with cornification or hypertrophic keratinized cells of the skin.

The oral mucosa also showed distinct hypertrophy in fetuses and the stratified squamous surface was continuous across the mouth. This continuity might explain the exceptionally short MCJ: the skin would suddenly lose the underlying dermal papillae. However, the mouth was not fused by any adhering cornification. Likewise, without cornification, the approximation surfaces of the eyelids in fetuses are tightly attached until 14-15 weeks of gestation (almost 100 mm CRL)^{[53, 54]}. but blinking begins in utero ^[5]. Suckling movements also start in utero ^{[55, 56]} and adhesion of the mouth seems to be avoided. The skin and MCJ generally contained abundant CD1apositive Langerhans cells, while the lamina propria of the mucosa usually contained abundant CD8-positive suppressive T lymphocytes.

At and near the MCJ epithelium, Langerhans cells seemed to constitute a limited subpopulation for receiving and presenting surface antigens. The skin and MCJ contained corpuscle-like nerve terminals along the wavy base of the epidermis, while free nerve ends were evident beneath the mucosa. However, the oral mucosa contains encapsulated nerve terminals ^[57] and a Merkel cell-neurite complex ^[58]. MCJ expansion was evident in the nasal orifice in specimens from elderly individuals. Such an expanded MCJ may occur as a result of stimulation by dry and/or cool air and is usually termed “squamous metaplasia” as a precursor tissue of squamous carcinoma ^[59]. Likewise, in the anal canal, elongation of the anal MCJ was most likely to occur along the supero-inferior axis with dilated hemorrhoid veins beneath the epithelium: this change may result from stimulation by hard feces.

The MCJ at the vulva and prepuce was long and wide even in fetuses, and had a specifically thin stratified epithelium. Although we suspected that the morphology might be influenced by age-related hormonal conditions, we had no opportunity to observe the histology in young adults. The vulva and prepuce are also characterized by strong and specific expression of CK7, as is the case in the urogenital tract (see the Introduction). Therefore, the MCJ might develop from the urethral mucosa. Despite having a very thick stratified squamous epithelium, the vagina also expresses CK7. Study limitations Because of delayed fixation and long-term preservation, immunohistochemistry of human fetal specimens often yielded inconsistent results, including absence of any reactivity.

Moreover, in previous studies, the antibodies employed changed and differed year-by-year. This made it difficult to compare immunohistochemical results, especially for cytokeratin, among various research groups. A further study using specimens from a single well-fixed young cadaver will therefore be necessary for a comparison among the five MCJ sites. Acknowledgment We are grateful to the individuals who donated their bodies to Tokyo Dental College for research and education on human anatomy. We also express deep gratitude to the families who donated fetuses to Akita University School of Medicine. Author Contributions CRediT authorship contribution statement Kei Kitamura: Conceptualization; data curation; formalanalysis; writing-original draft. Yuki Sugiyama: Conceptualization; data curation; formalanalysis; writing-original draft.

Kotoko Imai: Conceptu-alization; formal analysis; methodology; writing-originaldraft. Tianyi Yang: For-mal analysis; project administration; validation; writing review and editing. Ryo Sekiya: Conceptualization; investigation; methodol-ogy; supervision; writing-review and editing. Kazuma Morita: Conceptualization; Data curation; methodology; supervision; writing-review and editing. Gen Murakami: Formal analysis;investigation; supervision; writing-original draft; writing-review and editing. Hitoshi Yamamoto: Data curation; investigation; visualization;writing-review and editing. Shin-ichi Abe: Data curation; investigation; visualization;writing-review and editing Declarations Conflict of interest: No conflicts of interest declared. Ethical approval: This study was conducted in accordance with the Declaration of Helsinki. The use of the study specimens was approved by the Ethics Committee of Tokyo Dental College (No. 922-2, 932-2).

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