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Embryology of Congenital Malformations

  • Dietrich KluthEmail author
  • Roman Metzger
Living reference work entry

Abstract

Today, the embryology of numerous congenital anomalies in humans is still a matter of speculation. This is due to a number of reasons which include:
  • Misconceptions and/or outdated theories concerning normal and abnormal embryology.

  • A shortage of study material (both normal and abnormal embryos).

  • A shortage of explanatory images of embryos and developing embryonic organs.

  • Difficulties in the interpretation of serial sections.

In recent years, a number of animal models had been established which helped to overcome the shortage of both, normal and abnormal embryos. However, a general agreement on when, why and how abnormal development takes place, still does not exist. As a result, many typical malformations are still not explained satisfactorily and pediatric surgeons of all specialties are still confused when they are confronted with the background of normal and abnormal embryologic development.

Keywords

Human birth defects Animal models Teratology Human embryology 

Misconceptions and/or Outdated Theories Concerning Normal and Abnormal Embryology

Our understanding of the normal and abnormal development of embryos is still influenced by two theories:

  1. (a)

    The “biogenetic law” after Haeckel (1975)

     
  2. (b)

    The theory of “Hemmungsmißbildungen” (Schwalbe 1906)

     

According to Haeckel’s “biogenetic law,” a human embryo recapitulates in its individual development (ontogeny) the morphology observed in all lifeforms (phylogeny). This means that during its development, an advanced species (a human embryo) seems to pass through stages represented by adult organisms of more primitive species (Gilbert 2003). This theory has been used to “bridge” gaps in the understanding of normal embryonic development and still has an impact on the nomenclature of embryonic organs. This explains why human embryos have “cloacas” like adult birds and “branchial” clefts like adult fish.

The term “Hemmungsmißbildung” stands for the theory that malformations actually represent “frozen” stages of normal embryonic development. This theory too has been used to “bridge” gaps in the understanding of normal embryonic development in a manner which could best describe as “reversed embryology.” As a result, our knowledge of normal embryology stems more from pathological-anatomic interpretations of observed malformations than from proper embryological studies. The theory of the “rotation of the gut” as a step in normal development is a perfect example for this misconception (Fig. 1a). Others are “failed fusion of the urethral folds” (hypospadia, Fig. 1b), “failed closure of the pleuroperitoneal canals” (congenital diaphragmatic hernia, Fig. 1c), or “persistent cloaca” (Fig. 1d).
Fig. 1

Theory of “Hemmungsmißbildungen”: typical examples. (a) Schematic drawing of the “rotation of the gut” (After McVay et al. 2007, with permission from Elsevier). Impaired rotation causes “malrotation.” (b) Schematic drawing of urethral development (Modified after Hamilton et al. 1962). Hypospadia as a result of impaired fusion of urethral folds (UG). AP anal pit, GP glans penis, UO urethral opening, US urethral sulcus, SS scrotal swelling. (c) Schematic drawing of diaphragmatic development (Modified after Bromann 1902): impaired closure of diaphragmatic membranes causes diaphragmatic hernia. AO aorta, E esophagus, L diaphragm derived from lateral body wall, PN phrenic nerve, P pericardium, PPM pleuroperitoneal membrane, VC vena cava, PPC pleuroperitoneal canal, M diaphragm derived from dorsal mesenterium. (d) Schematic drawing of cloacal development (Stephens 1963). Impaired formation of the urorectal septum causes anorectal malformations

A Shortage of Study Material (Both Normal and Abnormal Embryos)

Today, a growing number of animal models exist which allow embryological studies in various embryological fields. This includes studies in normal as well as in abnormal embryos. Especially for the studies of esophageal and anorectal malformations, a number of animal models had been established.

A Shortage of Explanatory Images of Embryos and Developing Embryonic Organs

Advanced technology in a number of fields gives much better insights into human development. This includes ultrasonography of fetuses as well as magnetic resonance imaging (MRI). For detailed embryological studies, scanning electron microscopy is still a very useful tool. Recently, STEDING published a scanning electron microscopic atlas of human embryos which provides detailed insights into normal human embryology (Steding 2009). Scanning electron microscopy is the perfect tool to document embryonic structures:

  1. (a)

    Serial sectioning of embryos and time-consuming three-dimensional reconstructions are not necessary.

     
  2. (b)

    The embryo can be studied in all three dimensions “online.”

     
  3. (c)
    The images and photographs are of superior quality (Fig. 2).
    Fig. 2

    Scanning electron microscopy (SEM) electron microscopy enables a wide range of magnification and a superior quality of photographs: perineal region of a female rat, ED 20. The highest magnification shows detailed structures on the cell surface (SEM Pictures © D. Kluth)

     

Difficulties in the Interpretation of Serial Sections

Although a number of specific tasks demand the serial section of embryos, the difficulties in the interpretation must not be underestimated. Three-dimensional (3D) reconstructions, although feasible, are tainted with a loss of information, not only caused by the sectioning itself but also by the use of 3D image software.

Animal Models Used for Applied Embryology

Over the last two decades. a number of animal models have been developed with the potential to gain a better understanding of the morphology of not only malformed but also normal embryos. These animal models can be grouped in five subgroups:

  1. 1.

    Embryos of different species for the study of normal embryology

     
  2. 2.

    Surgical models

     
  3. 3.

    Chemical models

     
  4. 4.

    Genetic models

     
  5. 5.

    “Spontaneous” malformations of unclear cause

     

Embryos of Different Species for the Study of Normal Embryology

Human embryos are rare. Human embryos displaying typical anomalies are extremely rare. Therefore it makes sense to study specific developmental processes in embryos of animals with humanlike abnormalities. However, in all cases of animal models, the detailed study of normal embryos of the same species is mandatory.

We used scanning electron microscopy (SEM) in chicken, rat, and murine embryos in order to study certain embryological processes of the normal embryology of the foregut, the hindgut, the midgut, the testicular descent, and the development of the external genitalia. The advantage of chicken embryos is the high availability at low costs. They are easily accessible in the eggshell, and further breeding is possible when the eggs are treated accordingly. Embryos of rats and mice can be obtained in comparable large numbers; however, local regulations may limit the usage of mammalian embryos.

  1. (a)

    The chicken embryo was used to study foregut development. The aim was to clarify whether lateral ridges occur in the developing foregut or not and, when present, if they fuse to form the tracheoesophageal septum (Kluth et al. 1987; Metzger et al. 2011a) (Fig. 3).

     
  2. (b)

    Rat embryos were used to study, i.e., developmental processes during testicular descent, to clarify if “rotation” takes place during gut development (Fig. 4a), to assess the question if “cloacas” actually exist in rat embryos, and how the differentiation of the developing hindgut takes place (Fiegel et al. 2011; Metzger et al. 2011b; Kluth et al. 2011a) (Fig. 4b).

     
  3. (c)

    Mouse embryos were studied in the SD-mouse model (Fig. 5). Here, normal and abnormal hindgut development was studied (Kluth et al. 1995a).

     
Fig. 3

Animal models: chicken model is used to study the normal development of the foregut. (a) Schematic drawing of the developing foregut (Modified after Gray and Skandalakis 1972c). Dotted arrows indicate the growth directions of the esophagus (gray) and trachea (white). The small arrow in between indicates the growth direction of the assumed septum. The thick arrows point to the lateral ridges (insert) which are thought to appear in the foregut and which form the epithelial tracheoesophageal septum. (b) Schematic picture of the “water tap theory.” The lung anlage forms as a diverticulum which grows caudal and thus forms the trachea (Drawing modified after Merei and Hutson 2002)

Fig. 4

Animal models: rat embryos were used to study midgut development (a) and hindgut development (b) (SEM Pictures © D. Kluth)

Fig. 5

Animal models: SD-mice were used to study anorectal malformations. (a) Notice the short tail in a heterocygotic SD mouse. (b) Histology of the pelvic organs in a newborn heterozygous SD-mouse. The features of an anorectal malformation with rectourethral fistula (F) and a blind ending rectal pouch (RP) are detectable. U urethra. (c) The spectrum of malformations seen in SD-mice

Surgical Models

In the past, the chicken was an important surgical model to study embryological processes. As mentioned above, the easy access to the embryo, its broad availability, and its cheapness make it an ideal model for experimental studies. It has been widely used by embryologist especially in the field of epithelial/mesenchymal interactions (Goldin and Opperman 1980; Steding 1967; Jacob 1971). Pediatric surgeons have used this model to study morphological processes involved in intestinal atresia formation (Molenaar and Tibboel 1982; Schoenberg and Kluth 2002), gastroschisis (Aktug et al. 1997), and Hirschsprung’s disease (Meijers et al. 1992). The Czech embryologist Lemez (1980) used chicken embryos in order to induce tracheal agenesis with tracheoesophageal fistula (Fig. 6).
Fig. 6

Animal models: experimental embryology in chicken embryos. Metal clips were used to induce tracheal atresia (Lemez 1980). (a) Schematic drawing of the technique, (b) arrows indicate the area where the clips were positioned (SEM picture of a chicken embryo). (SEM Picture and schematic drawing © Dietrich Kluth)

Apart from these purely embryonic models, a large number of fetal models had been developed in the last 30 years. Although they were mainly created to study the feasibility of fetal interventions (Harrison et al. 1980), they also contributed to our current knowledge of normal and abnormal fetal development and fetal organ systems.

Chemical Models

It is well known that a number of chemicals (drugs, chemical fertilizers) can alter normal development of humans and animals alike. Some of these had been used to induce malformations similar to those found in humans. Most important today are:

  1. (a)

    The adriamycin model (Thompson et al. 1978; Diez-Pardo et al. 1996; Beasley et al. 2000)

     
  2. (b)

    Etretinate (Kubota et al. 1998; Liu et al. 2003)

     
  3. (c)

    All-trans-retinoic acid (ATRA) (Bitoh et al. 2002; Hashimoto et al. 2002; Sasaki et al. 2004)

     
  4. (d)

    Ethylenethiourea (Arana et al. 2001; Qi et al. 2002)

     
  5. (e)

    Nitrofen (Ambrose et al. 1971; Tenbrinck et al. 1990; Kluth et al. 1990; Costlow and Manson 1981; Irtani 1984)

     
  6. (f)

    Suramin and trypan (Männer and Kluth 2003, 2005)

     

Models (a–d) have been used to study atresia formation in the esophagus, the midgut, and the anorectum. Model (e) was developed to study malformations of the diaphragm, the lungs, the heart, and kidneys (hydronephrosis). Model (f) was used in chicken embryos to study the formation of cloacal exstrophy.

We used the nitrofen model to study the morphology of diaphragmatic hernia formation in rat embryos (Fig. 7).
Fig. 7

Animal models: the nitrofen model of diaphragmatic hernia. (a) Newborn rat with diaphragmatic hernia after nitrofen exposure at day 11.5. (b) Results of nitrofen exposure on days 9.5, 10.5, 11.5, 12.5, and 13.5. Most hernias were seen after nitrofen exposure on day 11.5

Genetic Models

Many aspects make genetic models the ideal model for the studies of abnormal development. In the past, a number of genetic models have been used for embryological studies of malformations. While older models were mostly the product of spontaneous mutations, newer models are, in most instances, the result of genetic manipulations mainly in mice (transgenic mice). The following models have been used by pediatric surgeons:

  1. (a)

    Models of spontaneous origin: The SD-mouse model (Dunn et al. 1940; Kluth et al. 1991). In the SD-mouse model, anorectal malformations are combined with anomalies of the kidneys, the spine, and the external genitalia (Fig. 5).

     
  2. (b)

    Inheritance models: the pig model of anal atresia (van der Putte and Neeteson 1984; Lambrecht and Lierse 1987) (Fig. 9). In pigs, anorectal malformations are seen quite frequently. One out of 300 newborn piglets present with anorectal malformations without evidence of genetical alterations.

     
  3. (c)

    “Knockout” models.

     
  4. (d)

    Viral models.

     
The number of transgenic animal models is currently growing fast. For pediatric surgeons those models are of major importance, which result in abnormalities of the fore- and hindgut. Here, interference with the Sonic hedgehog (Shh) pathway has proven to be very effective (Litingtung et al. 1998; Kim et al. 2001; Mo et al. 2001). There are two ways to interfere with that pathway:
  1. I.

    Targeted deletion of Sonic hedgehog (Litingtung et al. 1998; Kim et al. 2001)

     
  2. II.

    Deletion of one of the three transcription factors Glil, Gli2, and Gli3 (Kim et al. 2001; Mo et al. 2001)

     

It has been demonstrated, that targeted deletion of Sonic hedgehog resulted in homozygous Shh null mutant mice in the formation of foregut malformations like esophageal atresia/stenosis, tracheoesophageal fistulas, and tracheal/lung anomalies (Litingtung et al. 1998). In the hindgut, the deletion of Sonic hedgehog caused the formation of “cloacas” (Kim et al. 2001), while Gli2 mutant mice presented with the “classic” form of anorectal malformations and Gli3 mutants showed minor forms like anal stenosis (Kim et al. 2001; Mo et al. 2001). Interestingly, the morphology of Gli2 mutant mice embryos resembles that of heterozygous SD-mice embryos, while Shh null mutant mice embryos had morphological similarities with homozygous SD-mice embryos. It is interesting to note that after administration of adriamycin, abnormal pattern of Shh distribution could be demonstrated in the developing foregut (Arsic et al. 2004).

Recently, Botham et al. studied developmental disorders of the duodenum in mutations of the fibroblast growth factor receptor 2 gene (Fgfr2IIIb) (Botham et al. 2012). They noted an increased apoptotic activity in the duodenal epithelium of Fgfr2IIIb -/- embryos at day 10.5, followed by a disappearance of the endoderm at day 11.5. Interestingly, the duodenal mesoderm also disappeared within 2 days, and an atresia was formed. Similar processes had been observed in newborn piglets whose esophageal epithelium was removed via endoscopy (Booß and Okmian 1974; Komfält and Okmian 1973). This procedure resulted in esophageal atresias in these piglets.

In humans, viral infections are known to cause malformations. Animal models that use viral infections important for pediatric surgeons are very rare. One exception is the murine model of extrahepatic biliary atresia (EHBA) (Petersen et al. 1997). In this model, newborn Balb/c mice are infected with rhesus rotavirus group A45. As a result, the full spectrum of EHBA develops as it is seen in newborn with this disease. However, this model is not a model to mimic failed embryology. But it highlights the possibility that malformations are not caused by embryonic disorders but caused by fetal or even postnatal catastrophes.

Spontaneous Malformations Without Genetic Background

In chicken embryos, a number of spontaneous malformations can be observed. It is not quite clear which processes cause them. One reason may be a prolonged storage (more than 3 days) in fridges below 8 °C before breeding is started (Sydow H, Göttingen, Germany, “personal communication”). Spontaneous malformations of the head anlage (i.e., double anlage of the head, Fig. 8), the anlage of the heart, as well as abnormalities of the embryonic position (heterotaxia) are frequently seen (Sydow H, Göttingen, Germany, “personal communication”) (Fig. 9).
Fig. 8

Spontaneous malformation seen in a chicken embryo: double anlage of the head fold (Picture courtesy of H. Sydow, Göttingen, Germany)

Fig. 9

Animal models: a newborn piglet with anorectal malformation (Picture courtesy of W. Lambrecht, Hamburg, Germany)

This part on embryology and animal models highlights not only the importance to study embryos with experimental malformations but also the study of normal animal embryos. Today, much information in current textbooks on human embryology stems actually from studies done in animals of varies species. Many of these are outdated. The wide use of transgenic mice in order to mimic congenital malformations makes morphological studies of the organ systems in normal mice mandatory. Otherwise the interpretation of the effects by deletion of genetic information can be very difficult or even misleading.

Scanning Electron Microscopic Atlas of Normal and Abnormal Development in Embryos

In this section we want to present examples of normal and abnormal development as we have seen them in our studies in our labs over the past 30 years using scanning electron microscopy (SEM). We use the form of an embryological atlas following the old motto “A picture says more than a thousand words.” We focus on the following developmental processes:

  1. 1.

    Normal and abnormal foregut development (chicken embryos)

     
  2. 2.

    Normal and abnormal development of the diaphragm (rat embryos)

     
  3. 3.

    Development of the midgut (rat embryos)

     
  4. 4.

    Normal and abnormal development of the hindgut (mice and rats)

     
  5. 5.

    The development of the external genitalia and the urethra (rat embryos)

     
  6. 6.

    Testicular descent (rat embryos)

     

Foregut Development

Normal Foregut Development

Traditionally, foregut malformations like esophageal atresias and tracheoesophageal fistulas are explained by a faulty formation of the so-called “tracheoesophageal septum.” It is believed that normal septation takes place in two steps:
  1. I.

    Lateral endodermal ridges appear in the primitive foregut which fuse and form the tracheoesophageal septum.

     
  2. II.

    This solid endodermal septum is partly removed by apoptosis and substituted by mesenchymal cells (Fig. 3).

     

This theory had been described in detail by Rosenthal (1931) and Smith (1957). However, neither Zaw Tun (1982) nor O’Rahilly and Müller (1984) were able to confirm these sequences of embryological events. According to them, the term “separation” is a misnomer as the formation of the trachea is simply the result of the downgrowth of the respiratory diverticulum (Fig. 3) (Merei and Hutson 2002).

Using SEM, we studied the normal development of the foregut in chicken embryos (Kluth et al. 1987; Metzger et al. 2011a; Kluth and Habenicht 1987).

The first goal of these studies was to see if lateral endodermal ridges appear inside the foregut and if they fuse (Fig. 10). However, in our studies we were unable to identify ridges in the lateral foregut wall. Furthermore, signs of fusions of lateral foregut components were also not seen. As a reference we, added SEM pictures of the outflow tract in chicken hearts (pictures 10e, f courtesy of G. STEDING, Göttingen, Germany). Here, ridges appear which fuse and form the septa of the conus and the truncus. Note, that a line of fusion can be seen as an indicator that fusion actually took place. As no signs of fusion can be demonstrated in the foregut, theories dealing with improper formations of the tracheoesophageal septum are obsolete (Zaw Tun 1982).
Fig. 10

Embryology of the esophagus: SEM studies in chicken embryos. (a) The foregut of a chicken embryo of stage20/21, 3.5 days old. (b) The foregut is opened from lateral. The inner surface of the foregut is seen. Notice the absence of lateral folds (arrows). ES esophagus, TR trachea, * = tip of the tracheoesophageal fold. (c) View into the foregut from cranial. The tip of the tracheoesophageal fold can be seen. Notice the absence of fusion (higher magnification in (d). ES esophagus, TR trachea. (e) Process of fusion in the outflow tract of an embryonic heart (chicken embryo). Cushions in the outflow tract of the heart (c) fuse to form a septum. (f) Notice the fusion line which can be seen in an older embryo (arrows) (Pictures 10 e, f courtesy of G. Steding, Göttingen, Germany). SEM Pictures 10a–d © Dietrich Kluth

The second goal was to visualize the early formation of the lung bud (Fig. 11). In our series of embryos, we could demonstrate that after the formation of the early lung anlage, two lung buds appear, which are the forerunners of the bronchi. The anlage of the trachea itself is seen later as the floor of a “common foregut” chamber (Metzger et al. 2011a). Thus, not the trachea but the bronchi are the first organs of the respiratory tree that develop. This speaks against the idea of a simple downgrowth of the tracheal anlage as assumed by Zaw Tun and O’Rahilly and Müller (Zaw Tun 1982; O’Rahilly and Muller 1984).
Fig. 11

Embryology of the esophagus: formation of the respiratory tract. (a) Lung buds are the forerunners of the bronchi (LB). CF common space of the foregut. (b) The bronchi start to develop (L Br). A trachea is not visible yet. CF common space of the foregut. (c) The trachea (Tr) is still part of the common foregut. LaF larynx anlage, ES esophagus, L Br bronchi, St stomach, ** = fold which marks the border between the pharynx and esophagus. (SEM Pictures © Dietrich Kluth)

The third goal was to identify possible mechanisms of differentiation of the foregut into the larynx, pharynx, trachea, and esophagus. In our embryos, we could identify typical markers in the foregut (Fig. 12). In the dorsal aspect of the foregut, a fold appears which marks the borderline between the pharynx and esophagus. Cranially the larynx develops, and caudally, a fold appears between the developing trachea and the esophagus. In the next developmental steps, these folds approach each other but do not fuse. As a result, the area of the common foregut is reduced in size and later forms the pharyngo-tracheal canal (Kluth et al. 1987).
Fig. 12

Embryology of the esophagus: the common space of the foregut is reduced in size by a system of folds. (a) The trachea is still part of the common space (CF). LaF Larynx anlage. (b) The size of the CF common foregut is reduced by the growth of folds, which are formed by the larynx fold (LaF) from cranial, the tracheoesophageal fold (*) from caudal, and the fold between the pharynx and esophagus (**) from dorsal. (SEM Picture and schematic drawing © Dietrich Kluth)

The formation of esophageal atresia (Fig. 13)
Fig. 13

Embryology of the esophagus: hypothetical formation of foregut malformations. (a) Normal foregut of a chicken embryo, view from lateral into the foregut. Notice the reduced size of the common foregut space (***) due to the development of the folds. La Larynx. (b) Chicken embryo with a spontaneous foregut malformation. The pharynx ends blindly. Part of the trachea is in normal position and of tracheal size. The dorsal part of the common foregut space is missing (*). (c) Hypothetical explanation of foregut maldevelopment. (C1) The dorsal fold (*) between the pharynx and larynx grows too deep into the common foregut space. Consequently the rest of the common space develops into trachea, and an esophageal atresia with lower fistula develops. (C2) The common foregut space is reduced in size from ventral (*). Consequently the rest of the common space develops into esophagus, and a tracheal atresia with fistula occurs (very rare). (C3) Impaired development of the dorsal fold and the tracheoesophageal fold leads to an undivided common foregut space and a laryngo-tracheo-esophageal cleft. (SEM Picture and schematic drawing © Dietrich Kluth)

Although a number of models for abnormal foregut development exist, a clear morphological description of the embryological events that finally lead to esophageal atresias is still missing. Based on our observations, the development of the malformation can be explained by disorders either of the formation of the folds or of their developmental movements (Kluth et al. 1987; Metzger et al. 2011a; Kluth and Habenicht 1987):

  1. (a)

    Atresia of the esophagus with fistula (Fig. 13C1):

    The dorsal fold of the foregut bends too far ventrally. As a result the descent of the larynx is blocked. Therefore the common tracheoesophageal space remains partly undivided and lies in a ventral position. Due to this ventral position, the common space differentiates into trachea.

     
  2. (b)

    Atresia of the trachea with fistula (Fig. 13C2): The foregut is deformed on its ventral side. The developmental movements of the folds are disturbed, and the tracheoesophageal space is dislocated in a dorsal direction, where it differentiates into esophagus.

     
  3. (c)

    Laryngo-tracheo-esophageal clefts (Fig. 13C3): Faulty growth of the folds results in the persistence of the primitive tracheoesophageal space.

     

In our collection of chicken embryos, we came across an embryo with abnormal foregut features (Fig. 13b). When compared to normal embryos of the same age group (Fig. 13a), the following statements can be made: (I) Obviously, the pharynx ends blindly. (II) The dorsal part of the common foregut space is missing. (III) The ventral part of the common space has the size of a trachea. (IV) This foregut looks like the hypothetical form C1 in Fig. 13.

Development of the Diaphragm

Normal Development

The traditional theories of diaphragmatic development have been summarized by Kluth et al. (1989). Using SEM, we have recently restudied the diaphragmatic development. For practical reasons it is essential to note that the early diaphragm consists of two parts:

  1. (a)

    The septum transversum which, in young embryos, is identical to the floor of the pericardium

     
  2. (b)
    The structures that surround the pleural cavity. They are:
    1. I.

      The posthepatic mesenchymal plate (PHMP) (Irtani 1984), which covers the dorsal aspect of the liver and is in continuity to the septum transversum ventrally and cranially.

       
    2. II.

      The pleuroperitoneal fold (PPF) which separated the pleura from the peritoneal cavity. This fold connects ventrally to the septum transversum and the PHMP and dorsally to the mesonephric ridge (Mayer et al. 2011). This PPF is a structure that is identical to the pleuroperitoneal membrane of the old literature (Kluth et al. 1989).

       
    3. III.

      The dorsal mediastinum which contains the esophagus, the trachea, and the aorta.

       
     
According to our SEM studies, the PHMP plays the most important role in normal diaphragmatic development. In Figs. 14 and 15, the closure process of the pleuroperitoneal openings (PPO) is depicted. At embryonic day (ED) 13, the formation of the PHMP and its lower border can be seen (Fig. 14a). The PHMP then expands dorsolaterally at embryonic day 13.5 (Fig. 14b), establishing a new lower border.
Fig. 14

Normal development of the diaphragm: caudal growth of the posthepatic mesenchymal plate (PHMP) (Irtani 1984). (a) Rat embryo, ED 13. View at the dorsal part of the diaphragm. The dorsal diaphragm is short. The black line marks the caudal border of the PHMP. Arrows indicate the direction of future PHMP growth. Note the large area of the liver still uncovered by the PHMP. (b) Rat embryo 13.5 days. Note the caudal growth of the PHMP within 12 h (second dark line). The uncovered liver is markedly smaller. (SEM Pictures © Dietrich Kluth)

Fig. 15

Normal development of the diaphragm: closure of the pleuroperitoneal openings (PPO). Rat ED 15 (a) and ED 16 (b). Most of the liver (Li) is covered by the posthepatic mesenchymal plate (PHMP). At ED 16 the only intra-abdominal organ seen is the tip of the gonads (Go). (SEM Pictures © Dietrich Kluth)

In Fig. 15, the final closure of the PPO is shown. In this process the PHMP starts to cover the last free areas of the liver (Fig. 15a). In this process, the pleuroperitoneal fold (PPF) plays only a minor role.

In the literature, the nomenclature of the various parts of the diaphragm is confusing. We use the term PPF for a structure which was formally known as pleuroperitoneal membrane (Kluth et al. 1989; Mayer et al. 2011). The term PPF is used differently by Greer and coworkers (Clugston et al. 2010). Their PPF is very similar to the PHMP as described by IRITANI and us but seems to include the ventral part of our PPF.

Abnormal Development

In the past, several theories were proposed to explain the appearance of posterolateral diaphragmatic defects:

  1. (a)

    Defects caused by improper development of the pleuroperitoneal membrane (Grosser and Ortmann 1970; Gray and Skandalakis 1972a; Holder and Ashcraft 1979)

     
  2. (b)

    Failure of muscularization of the lumbocostal trigone and pleuroperitoneal canal, resulting in a “weak” part of the diaphragm (Gray and Skandalakis 1972a; Holder and Ashcraft 1979)

     
  3. (c)

    Pushing of the intestine through posterolateral part (foramen of Bochdalek) of the diaphragm (Bremer 1943)

     
  4. (d)

    Premature return of the intestines into the abdominal cavity with the canal still open (Gray and Skandalakis 1972a; Holder and Ashcraft 1979)

     
  5. (e)

    Abnormal persistence of the lung in the pleuroperitoneal canal, preventing proper closure of the canal (Gattone and Morse 1982)

     
  6. (f)

    Abnormal development of the early lung and posthepatic mesenchyme, causing non-closure of pleuroperitoneal canals (Irtani 1984)

     

Of these theories, failure of the pleuroperitoneal membrane to meet the transverse septum is the most popular hypothesis to explain diaphragmatic herniation. However, using SEM techniques (Kluth et al. 1989; Mayer et al. 2011), we could not demonstrate the importance of the pleuroperitoneal membrane for the closure of the so-called pleuroperitoneal canals (Fig. 15).

It is still speculated that delayed or inhibited closure of the diaphragm will result in a diaphragmatic defect that would allow herniation of the gut into the fetal thoracic cavity. In a series of normal staged embryos, we measured the width of the pleuroperitoneal openings and the transverse diameter of gut loops (Kluth et al. 1995a). On the basis of these measurements, we estimated that a single embryonic gut loop requires at least an opening of 450 μm size to herniate into the fetal pleural cavity. However, in none of our embryos, the observed pleuroperitoneal openings were of appropriate dimensions. This means that delayed or inhibited closure of the pleuroperitoneal canal cannot result in a diaphragmatic defect of sufficient size. Herniation of the gut through these openings is therefore impossible. Thus the proposed theory about the pathogenetic mechanisms of congenital diaphragmatic hernia (CDH) development lacks any embryological evidence. Furthermore the proposed timing of this process is highly questionable (Kluth 1993).

Animal Model

An animal model for diaphragmatic hernia has been developed (Ambrose et al. 1971; Tenbrinck et al. 1990; Kluth et al. 1990; Costlow and Manson 1981; Irtani 1984) using nitrofen as noxious substance. In these experiments CDHs were produced in a reasonably high percentage of newborns (Kluth et al. 1990). We collected a number of affected embryos of different age groups and studied these using SEM (Kluth and Tander 1995; Kluth 1993). Our results (Figs. 16 and 17) are as follows:
  1. (a)

    Timing of diaphragmatic defect appearance. Iritani (Irtani 1984) was the first to notice that nitrofen-induced diaphragmatic hernias in mice are not caused by an improper closure of the pleuroperitoneal openings but rather the result of a defective development of the posthepatic mesenchymal plate (PHMP). In our study in rats, clear evidence of disturbed development of the diaphragmatic anlage was seen on ED 13 on the left and ED 14 on the right diaphragm anlage (Fig. 14) (Kluth and Tander 1995; Kluth et al. 1996). In all embryos affected, the PHMP was too short. This age group is equivalent to 4–5 week-old human embryos (Kluth and Tander 1995).

     
  2. (b)

    Location of diaphragmatic defect. In our SEM study, the observed defects were localized in the area of the PHMP (Fig. 14). We identified two distinct types of defects (Haeckel 1975): large “dorsal” defects and (Schwalbe 1906) small “central” defects (Kluth and Tander 1995). Large defects extended into the region of the pleuroperitoneal openings. In these cases, the closure of the pleuroperitoneal openings was usually impaired by the massive ingrowth of the liver (Figs. 14 and 15). If the defects were small, they were consistently isolated from the pleuroperitoneal openings which closed normally at the 16th or 17th day of gestation. Thus, in our embryos with CDH, the region of the diaphragmatic defect was a distinct entity and was separated from that part of the diaphragm where the pleuroperitoneal “canals” are localized. We conclude therefore that the pleuroperitoneal openings are not the precursors of the diaphragmatic defect.

     
  3. (c)

    Why lungs are hypoplastic. Soon after the onset of the defect in the 14-day-old embryo, the liver grows through the diaphragmatic defect into the thoracic cavity (Fig. 14). This indicates that from this time on, the available thoracic space is reduced for the lung, and further lung growth is hampered. In the following stages, up to two- thirds of the thoracic cavity can be occupied by the liver (Figs. 16 and 17). Herniated gut was found in our embryos and fetuses only in late stages of development (21 days and newborns) (Fig. 17). In all of these, the lungs were already hypoplastic, when the bowel entered the thoracic cavity (Kluth and Tander 1995).

     
Fig. 16

Dorsal diaphragms after nitrofen exposure at rat ED 11.5. (a, b) Rat embryo at ED 14. The abnormal anlage of the right diaphragm is easy to see. Dotted arrows mark the diameter of the uncovered liver (Li). On the left, the development of the posthepatic mesenchymal plate (PHMP) is normal. On the right, the PHMP stopped to grow to caudal. (c) Rat ED 17. A small hernia (liver) can be seen. Note the position close to the vena cava (large arrow). The small arrow points to the closed pleuroperitoneal openings (PPO). (d) Rat ED 18. The hernia is big. Two lobes of the liver project into the thorax. The big arrow points to the vena cava. Small arrows mark the border of the PPO, which is open due to the ingrowth of the liver. Note that the size of the diaphragmatic defect is much larger than the PPO itself. (SEM Pictures © Dietrich Kluth)

Fig. 17

Huge hernias after nitrofen exposure. (a) Rat ED 20. In none of our embryos, the gut could be found in this age group. (b) Rat ED 21. In this age group and older animals, the gut can be found inside the thoracic cavity. (SEM Pictures © Dietrich Kluth)

Based on these observations, we conclude that the early ingrowth of the liver through the diaphragmatic defect is the crucial step in the pathogenesis of lung hypoplasia in CDH. This indicates that growth impairment is not the result of lung compression in the fetus but rather the result of growth competition in the embryo: the liver that grows faster than the lung reduces the available thoracic space. If the remaining space is too small, pulmonary hypoplasia will result.

Development of the Hindgut

Normal Development

As in the foregut, a process of septation has been postulated for the proper subdivision of the “cloaca” into the dorsal anorectum and the ventral sinus urogenitalis. Disorders in this process of differentiation are thought to be the cause of cloacal anomalies such as persistent cloaca and anorectal malformations (Stephens 1963).

However, for many years, this process of septation has been under dispute. Some authors (Toumeux 1888; DeVries and Friedland 1974) believe that the descent of a single fold separates the urogenital part from the rectal part by ingrowth of mesenchyme. Others (Retterer 1890) think that lateral ridges appear in the lumen of the cloaca, which progressively fuse along the midline and thus form the septum. Van de Putte (vd Putte 1986) denied the existence of any process of septation.

In the recent years we studied the cloacal development in rats and SD-mice embryos using SEM techniques (Kluth et al. 1995b, 2011a; Kluth and Lambrecht 1997).

The first goal of these studies was to see if lateral ridges appear inside the “cloaca” and if these actually fuse to form an endodermal septum (Fig. 18). As in the foregut of chick embryos, we were unable to see lateral ridges (Fig. 18c) projecting into the cloacal lumen. Signs of median fusion of lateral cloacal parts were also lacking (Fig. 18a, b). However, in contrast to Van de Putte (vd Putte 1986), our SEM studies indicate that downgrowth of the tip of the urorectal fold takes place (Fig. 19a, b), although it is probably not responsible for the formation of cloacal malformations.
Fig. 18

Normal development of the hindgut. Rat ED 14. (a) The ventral part of the cloaca is removed. As in the foregut, signs of fusion are lacking. (b) Schematic drawing of the situation in (a). (c) After removal of the lateral wall of the cloaca, internal ridges which could form an urorectal septum are not detectable. (SEM Pictures and schematic drawing © Dietrich Kluth)

Fig. 19

Normal development of the hindgut. a, b The “cloaca” (c) in a rat embryo ED 14.5 has the following features: proximal urethra (PU), distal urethra (DU), rectum (HG), tail gut (TG), cloacal membrane. The line marks the border between the rectum and urethra. The schematic drawing shows the situation in a rat embryo ED 14. Note that the “cloaca” is not equally divided by the line. The gray area in the schematic drawing marks the area of the future anus. It lies in the dorsal part of the cloacal membrane close to the tail fold. (SEM Pictures and schematic drawing © Dietrich Kluth)

Our findings on normal embryology of the hindgut were:
  1. (a)

    The “cloaca” is not subdivided into two equal parts (Fig. 19a, b). The much larger ventral part gives rise to the future distal urethra.

     
  2. (b)

    The dorsal “cloaca” contains the future anorectal region. The future anal opening is situated in the dorsal part of the “cloacal’ membrane,” close to the tail fold (Fig. 19b).

     

Abnormal Development

As already mentioned, a number of animal models exist which allow embryological studies of abnormal hindgut development. In our studies we used embryos obtained from SD-mice. The SD-mouse is a spontaneous mutation of the house mouse, characterized by a short tail (Fig. 5). Homozygous or heterozygous offspring of these mice shows skeletal, urogenital, and anorectal malformations (Kluth et al. 1991). Therefore these animals are ideal for detailed studies of anorectal malformations.

In all affected embryos, we made the following observations (Fig. 20a–d):
  1. (a)

    Compared to normal embryos (Fig. 20a, b), we found abnormally shaped cloacas. The dorsal part was always missing (Fig. 20c, d).

     
  2. (b)

    The cloacal membrane was always too short (Fig. 20c, d). In all cases the dorsal part of the cloacal membrane was absent.

     
  3. (c)

    The proximal hindgut joined the cloaca at an abnormal position (Fig. 20c, d).

     
Fig. 20

Normal (a) and abnormal hindgut (c) in heterozygous SD-mouse embryos. Note that in the abnormal hindgut, the dorsal cloaca, which contains the area of the future anus, is completely missing. As a result, the rectum keeps in contact with the urethra too high (so-called fistula). The cloacal membrane (CM) is too short. In (b, d) the findings are summarized in schematic drawings. A future bladder, U ureter, W WOLFF duct, HG rectum (hindgut), C “cloaca,” TG tail gut. (SEM Pictures and schematic drawing © Dietrich Kluth)

Figure 21 summarizes the developmental processes in a sketch.
Fig. 21

Hypothetical line of events in anorectal malformations. (a) In young embryos, the cloacal membrane is too short. (b) As a result, the dorsal part of the “cloaca” is missing, which normally contains the area of the future anal canal. (c) The rectum remains attached to the future urethra. (Schematic drawing © Dietrich Kluth)

Development of the External Genitalia and the Urethra

Many investigators (Felix 1911; Spaulding 1921; Glenister 1958) believe that the urethra develops by fusion of the paired urethral folds (Fig. 22) which takes place following the disintegration (rupture) of the ventral part of the cloacal membrane, the so-called “urogenital membrane.” Impairment of this process of fusion is thought to result in the different forms of hypospadias (Gray and Skandalakis 1972b). In order to get more information about this process, we studied the formation of the external genitalia in staged rat embryos and fetuses (Kluth et al. 1988, 2011b).
Fig. 22

Normal genital development. It is a common assumption that the “cloacal membrane” ruptures not only in the dorsal (anal) part but also in the ventral (urethral) part. This would lead to a situation as depicted in (a). AP anal pit, GP glans penis, UO urethral opening, US urethral sulcus, SS scrotal swelling. In (b) the phallus of a normal rat embryo is shown. In this age group (ED 17,5), the sex of the embryo cannot be estimated by the appearance of the outer genitals. (SEM Picture © Dietrich Kluth)

Normal Development of the External Genitalia

This study was carried out in normal rat embryos and fetuses between embryonic day 17.5 (Fig. 22) and embryonic day 20 (Fig. 24).
  1. (a)

    Rupture of the cloacal membrane (Fig. 23).

    At embryonic day 17.5, the dorsal disintegration of the cloacal membrane can be seen (Fig. 23a, b). The ventral (urethral) part of the cloacal membrane remains intact. In Fig. 23c, this process of disintegration is seen in more detail. The ectodermal part of the cloacal membrane shows clear signs of disintegration. The tip of the urorectal fold is seen which later forms the perineum. Ventral to the tip of the urorectal fold, an opening is seen which is in connection to the distal urethra. Dorsally the anal opening is seen. At this time point, the external genitals allow no differentiation between the sexes.

     
  2. (b)

    Further development of the external genitalia.

    A rupture of the ventral part of the cloacal membrane cannot be seen in older embryos and fetuses. In males, the transient urethral opening disappears. Later a “raphe” is seen in this position (Fig. 24a). In females, this “raphe” is missing (Fig. 24b).

     
  3. (c)
    Special dissections of a rat embryo at embryonic day 18.5 allow the following statements (Fig. 25a–c): The urethra is composed of two parts, the proximal and the distal part. The epithelium of the distal part reaches to the tip of the phallus. It is interesting to see that the urethra is connected to the perineal region by a short canal, the so-called “cloacal canal.” In our opinion this is the future female urethra.
    Fig. 23

    Rupture of the dorsal cloacal membrane in a rat embryo (ED 17.5). (a, b) The rupture of the membrane is clearly seen. (c) In high magnification the situation is visible in detail. Half of the genitals is removed by micro preparations. The tip of the urorectal fold can be seen (URF). Ventrally the opening of the urethra is seen (UO). The rectum (Re) opens dorsally (AO). Fusion of the URF with the cloacal membrane, as assumed by some researchers, does not take place. EC ectoderm, CP cloacal plate, d Ur distal urethra, p Ur proximal urethra. (SEM Pictures © Dietrich Kluth)

    Fig. 24

    Rat fetuses ED 20, (a) male rat, (b) female rat. The sex is discernable by external inspection of the genitals. Note the “raphe” (*) in (a), which is typical for the male phallus. This raphe is not the result of fusion, as generally believed. In female rats this “raphe” is missing. (SEM Pictures © Dietrich Kluth)

    Fig. 25

    Phallus of a rat ED 18.5. (a) The sex is not discernable by external inspection. The lateral portion of the phallus and the lateral wall of the urethra are removed. p Ur proximal urethra, d UR distal urethra, Re rectum. (b) The sketch describes the situation in (a): p Ur proximal urethra, d UR distal urethra, CC urethral opening in females. Note that the urethra in this stage is not sexually determined. The female urethra is short and ends at the CC opening. (c) The male urethra is formed by the distal urethra, which extends to the tip of the phallus. Sy symphysis, Ur urethra, C cloacal membrane, Re rectum. (SEM Pictures and schematic drawings © Dietrich Kluth)

     
Summarizing our results we found:
  1. (a)

    In rats, the urethra is always present as a hollow organ during urethral embryogenesis and that it is always in contact with the tip of the genitals.

     
  2. (b)

    Initially a double urethral anlage exists. The differentiation in female and male urethra happens in rats more than 18.5 days old.

     
  3. (c)

    We had no evidence for the disintegration of the urogenital cloacal membrane and a fusion of lateral portions within the perineum.

     

Abnormal Development of the External Genitalia (Hypospadia Formation)

In our opinion, more than one embryological mechanism is at play in the formation of the hypospadia complex (Kluth et al. 1988, 2011a). The moderate degrees, such as the penile and glandular forms, represent a developmental arrest of the genitalia. They take their origin from a situation comparable to the 20-day-old embryo. Consequently the penis, not the urethra, is the primary organ of the malformation. Perineal and scrotal hypospadias are different from the type discussed previously. Pronounced signs of feminization in these forms suggest that we are dealing with a female-type urethra. Origin of this malformation complex is an undifferentiated stage as may be seen in the 18.5-day-old rat embryo.

The Development of the Midgut

Traditional Theories

Traditionally, the midgut development is described as a process of “rotation.” In this process the following parts are involved: the distal part of the duodenum, the small bowel, and most parts of the big bowel. The process of rotation takes place in two phases (Mall 1898; Frazer and Robbins 1915):
  1. (a)

    In the first phase, the midgut loop develops inside the umbilicus (so-called “physiological herniation of the midgut”). Here, a 90-anti-clockwise rotation around the axis of the mesentery is thought to take place.

     
  2. (b)

    After the “return” of the midgut into the abdominal cavity, another anti-clockwise “rotation” of 180° is thought to take place inside the abdominal cavity (second phase). As a result, the region of the cecum moves to the right, thus overcrossing the mesenteric root, while the flexura duodeno-jejunalis is pushed to the left beneath the root of the mesentery (see schematic drawing in Fig. 1b) (Mall 1898; Frazer and Robbins 1915). These two phases sum up 270°. In contrast to this description, Grob (1953) subdivides this intra-abdominal process of rotation into two steps of 90° each.

     

Own Observations

We studied midgut development in rat embryos using SEM (Figs. 26, 27, and 28) (Metzger 2011a; Kluth et al. 1995b, 2003). Starting at embryonic day 13, the following parts of the midgut loop can be seen (Fig. 26a):
  1. (a)

    A central part with the duodenum and the distal colon close to the root of the mesentery.

     
  2. (b)

    A ventral part inside the extra embryonic coelom of the umbilicus (so-called “physiological herniation”). Here the cecum and the distal small bowel can be identified.

     
  3. (c)

    A middle part which connects the central part with the ventral part inside the umbilicus. Here the umbilical vessel, the small bowel on the right, and the proximal part of the colon on the left can be seen (Figs. 26b and 27d).

     
Fig. 26

Normal development of the midgut. (a) Rat embryo ED 13. The early midgut consists of three parts: a central part with the primitive duodenal loop (du), an extraembryonal part in the extraembryonic sac of the umbilicus (ce), and a straight part in between (sb). (b, c) The development of the central part, the duodenal loop (du), is seen. Note that the duodenojejunal junction is pushed beneath the root of the mesentery (arrows in c). This is caused by longitudinal growth of the duodenum. sb mall bowel loops, li liver. (SEM Pictures © Dietrich Kluth)

Fig. 27

Normal development of the midgut. (a) Rat ED 13. The cecum and the most distal part of the small gut are seen in the extraembryonic sac of the umbilicus. Dotted arrows indicate the border between extraembryonic and intraembryonic coelom. (b, c) Rapid lengthening of the small bowel leads to the formation of loops inside the extarembryonic sac of the umbilicus. Arrows indicate the direction of growth. (d) Note that during this process, rotation around the axis of the mesentery does not take place. ce cecum, sb small bowel, co colon, mV mesenteric vessel. (SEM Pictures © Dietrich Kluth)

Fig. 28

Normal development of the midgut. Rat ED 17. (a) The cecum (ce) is found in the abdominal cavity. A small bowel loop is still outside in the umbilical sac (arrow). Co colon, du duodenum. (b) Higher magnification of the area of the ventral body wall. Ce cecum, co colon. (SEM Pictures © Dietrich Kluth)

In the further development (ED 14–16), growth activities are seen in the area of the duodenum and inside the extraembryonic coelom.

Figure 26 shows the steps important for the duodenal developmental. In Fig. 26b (rat embryonic day 15) the duodenojejunal loop has been formed due to longitudinal growth of the duodenum. Further growth pushes this loop beneath the root of the mesentery (Fig. 26c).

Figure 27 shows the development of the intra-umbilical loops. These loops are the result of longitudinal lengthening of the small gut. Note the absence of any signs of rotation around the axis of the mesentery in Fig. 27d in a phase of active loop development inside the extra embryonic coelom.

Figure 28 shows the “return” of the midgut into the abdominal cavity. The cecum is seen inside the abdominal cavity in a ventral position close to the abdominal wall (embryonic day 17). The colon is entirely to the left in this phase of development. It is a small bowel loop which is still extraembryonic inside the umbilicus. In this phase of small bowel “return,” the bowel loops have already developed locally inside the abdominal cavity (Fig. 26c).

We conclude from our observations that the midgut can be subdivided in three parts, of whom the central and the ventral parts are of major importance. Localized longitudinal growth in the area of the duodenum leads to the formation of the duodenojejunal loop and its final position beneath the root of the mesentery. At the same time, localized growth of the small bowel has led to the formation of bowel loops inside the umbilicus and, later, inside the abdominal cavity. The growth activity of the large bowel is minimal, compared to that of the small bowel. Neither in the phase of loop formation inside the extraembryonic coelom of the umbilicus nor in the phase following the “return” of the gut into the abdominal cavity rotation of the gut around the axis of the mesentery can be observed. All processes in midgut development are the result of longitudinal lengthening of gut.

Testicular Descent

Since John Hunter in 1762, many researchers studied the embryology of testicular descent. In many of these studies, the importance of the gubernaculum during this process has been highlighted. However, a clear illustration of this rather simple process is still lacking (Heyns and Hutson 1995).

Today, most researchers in the field (Heyns 1987; Hullinger and Wensing 1985; Wensing 1988; Hutson et al. 2015) see two developmental phases during testicular descent:
  1. (a)

    The intra-abdominal descent: In this phase, the testis, which initially lies in close contact to the kidney, moves into the inguinal area.

     
  2. (b)

    The inguinal descent: In this phase the testis moves into the area of the scrotum.

     
We (Fiegel et al. 2010, 2011) studied the morphology of testicular descent in rat embryos between embryonic day 15 and 20 using SEM in order to illustrate in detail the various steps of the testicular development. While in rat embryos at embryonic day 15 male and female gonads look still identical (Fig. 29a), they become clearly distinguishable in rats of embryonic day 16 (Fig. 29b). The male gonad is getting thicker and slightly shorter than the female gonad, but both gonads are initially in close approximation to the kidneys.
Fig. 29

Descensus of the testis. Rat ED 15, female rat in (a), male rat in (b). Notice the difference in the size of the male and female gonads (go). Both gonads lie in close approximation to the kidneys (ki). (SEM Pictures © Dietrich Kluth)

Starting at embryonic day 16.5, the testis moves away from the lower pole of the kidney. On ED 19 the testis is located between the lower kidney pole and the roof of the bladder (Fig. 30a) and moves toward the bladder neck at ED 21 (Fig. 30b). This brings the intra-abdominal descent to an end and the inguinal descent starts.
Fig. 30

Testicular descent: intra-abdominal descent (first phase). (a) Male rat, ED 19. The gonads (go) have lost contact to the lower pole of the kidneys (ki) and lie in the middle portion between the kidney and bladder (bl). (b) Male rat, ED 21. The gonads are now close to the bladder in the inguinal area. This movement relative to the urinary bladder cannot be attributed to the relatively ascent of the kidneys. (SEM Pictures © Dietrich Kluth)

At the end of the intra-abdominal descent (ED 22), the bulb of the gubernaculum is still visible. (Fig. 31a). A little later, around birth (embryonic day 22), the bulb disappears partially and the processus vaginalis peritonei (PVP) develops (compare with Fig. 31b). Notice the rest of the bulb at the lower pole of the PVP. In this phase, the corda of the gubernaculum is still visible and attached to the caudal part of the epididymis, which has entered the PVP. At birth and later, the testis finally enters the PVP (Fig. 31c).
Fig. 31

Testicular descent: inguinal descent (second phase). (a) Male rat ED 21. The testis (T) has reached a position close to the inguinal region. The bulbic gubernaculum (GB) is still present. BL bladder, AE epididymis. (b) Male newborn rat, D 0. The bulbic part of the gubernaculum (GB) disappeared, and the processus vaginalis peritonei is formed (PVP). The border between peritoneal cavity and PVP is marked by arrows. The epididymis has entered the PVP. The corda of the gubernaculum is still visible. (c) Male newborn D 1–5. Not only the epididymis but also half of the gonads (T) has entered the PVP. The gubernaculum has completely disappeared. Arrows mark the border between the peritoneal cavity and the PVP. AE epididymis. (SEM Pictures © Dietrich Kluth)

The Role of the Gubernaculum

In our series, we studied the formation and the fate of the gubernaculum (Fig. 32). In rat embryos at embryonic day 16, the gubernaculum consists of two parts, the gubernacular bulb and the corda of the gubernaculum (Fig. 32a). The corda is rather attached to the lower anlage of the epididymis (Fig. 32a–c) than to the testis, as it is often described in the literature. Furthermore, it is often assumed that the testis is pulled downward by corda and bulbus. While this – in initial stages – seems to be morphologically possible, we identified later stages, where the testis and gubernaculum were positioned in such a way that pulling of the testis by the gubernaculum seems to be impossible (Fig. 32c, e).
Fig. 32

Testicular descent: in this series of SEM pictures, the morphology of the gubernaculum is shown (rat ED 19). (a, b) Here the gubernaculum consist of two parts, the corda of the gubernaculum (arrows in a, b) and the bulbus of the gubernaculum (GB). The corda inserts rather into the caudal anlage of the epididymis (AE)/mesonephric ridge than into the testis (T), as often assumed. BL bladder. (c) While in (a, b) tension caused by the gubernaculum seems to be theoretically possible, (c) demonstrates that the testis (T) is rather blocked by the bulb of the gubernaculum (GB) than pulled. Rat ED 21, AE epididymis. (d) Many researchers believe that tension caused by the corda is at play during testicular descent. However, the morphology of the insertion zone of the corda into the anlage of the epididymis shown in (a, b) speaks against this assumption. Sketch on the left shows expected morphology (traction!) versus observed morphology on the right. AE epididymis, T testis, GB bulbus of the gubernaculum. (e) The sketch summarizes our morphological data on the developmental sequence of the testicular descent. (SEM Pictures and schematic drawings © Dietrich Kluth)

Thus in our findings, we cannot support the opinions about the role of the gubernaculum during the testicular descent. Its main role seems to be its transformation into the PVP. We believe that intra-abdominal pressure probably plays an active role at least in the phase of the inguinal phase of testicular descent.

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© Springer-Verlag GmbH Germany 2016

Authors and Affiliations

  1. 1.Department of Pediatric Surgery, University HospitalUniversity of LeipzigLeipzigGermany
  2. 2.Department of Paediatric and Adolescent SurgeryParacelsus Medical University SalzburgSalzburgAustria

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