Placentation in the eastern water skink (Eulamprus quoyii): a placentome-like structure in a lecithotrophic lizard

Abstract

The eastern water skink (Eulamprus quoyii) has lecithotrophic embryos and was previously described as having a simple Type I chorioallantoic placenta. Indeed, it was the species upon which the definition of a Type I placenta was thought to be based, although we had cause to question that assumption. Hence we have described the morphology of the uterus of E. quoyii and found it to be more complex than previously supposed. The mesometrial pole of the uterus in E. quoyii displays a vessel-dense elliptical structure (the VDE) with columnar uterine epithelial cells. As pregnancy proceeds, the uterine epithelium near the mesometrial pole becomes folded and glands become hypertrophied, so that the morphology of VDE resembles that of a placentome, characteristic of Type III placentae. Unlike species with a Type III placenta, the apposing chorioallantoic membrane of E. quoyii is lined with squamous cells and interdigitates with the folded uterine epithelium. The remainder of the uterus is thin with a squamous uterine epithelium throughout pregnancy. Immunohistochemical localisation of blood vessels reveals a dense network of small capillaries directly beneath the folded epithelium of the VDE, while blood vessels are larger and sparser at the abembryonic pole of the uterus. Alkaline phosphatase (AP) activity is present in the uterine epithelium and sub-epithelial blood vessels in newly ovulated females. AP activity disappears from the epithelium between stages 27 and 29 of embryonic development and from the blood vessels after stage 34, but appears in the uterine glands at stage 35, where it remains until the end of pregnancy. Although the VDE is structurally similar to the placentomes found in other viviparous lizards, different distributions of AP activity in the uterus of E. quoyii and Pseudemoia spenceri suggest that the VDE may be functionally different from the placentome of the latter species. Our description of uterine morphology in E. quoyii provides evidence that, at least in some lineages, the evolution of a placentome may not occur in concert with the evolution of microlecithal eggs and obligate placentotrophy.

Figure 1

Haematoxylin and eosin-stained paraffin sections of the uterus of pregnant Eulamprus quoyii. (A) Squamous uterine epithelial cells on the abembryonic side of the uterus containing a stage 28 embryo. Scale bar: 20 μm. (B) Cuboidal–columnar uterine epithelial cells near the mesometrial pole of the uterus in a female with newly ovulated eggs (embryonic stages ≤ 25). Scale bar: 20 μm. (C) Deep folds of the uterine epithelium form rectangular-shaped plateaus in the inter-embryonic segments of a uterus containing newly ovulated eggs (embryonic stages 0–25). Scale bar: 40 μm. (D) Lipid inclusions are visible in the lamina propria of the lateral region of the uterus containing a stage 28 embryo. Scale bar: 20 μm. E, uterine epithelium; ES, eggshell; G, uterine gland; M, muscle. Short, thick arrows indicate lipid inclusions in the lamina propria.

Figure 2

Haematoxylin and eosin-stained paraffin sections of the uterus of pregnant Eulamprus quoyii. (A) A uterus containing a stage 32 embryo exhibits bumps and folds in the uterine epithelium near, but not centered across, the mesometrial pole. Scale bar: 320 μm. (B) Uterine glands are hypertrophied underneath the folded columnar epithelium in a stage 32 specimen. Scale bar: 20 μm. (C) Uterine epithelium is columnar on top of the apex of each fold but becomes squamous between folds in a stage 32 specimen. Scale bar: 20 μm. (D) Columnar uterine epithelium without folds near the mesometrial pole of a stage 33 specimen. Short, thick arrows indicate secretory vesicles in the epithelium. Scale bar: 13 μm. (E) Columnar epithelial cells near the mesometrial pole in a stage 34 sample have not yet formed folds, but are rippled. Scale bar: 20 μm. (F) Squamous uterine epithelium in the abembryonic region of the uterus in a stage 34 specimen. Scale bar: 13 μm. Ab, Abembryonic region of the uterus; E, uterine epithelium; G, uterine gland; M, muscle; MP, mesometrial pole.

Figure 3

Haematoxylin and eosin-stained paraffin sections of the uterus of pregnant Eulamprus quoyii. (A) A hypertrophied gland lies underneath columnar uterine epithelium in the mesometrial region of a stage 37 specimen. Scale bar: 20 μm. (B) Hypertrophied glands are densely packed in the lamina propria of the uterus in the mesometrial region of a stage 37 specimen. Scale bar: 20 μm. (C) The folded epithelium forms lobes separated by attenuated squamous epithelium in the mesometrial uterine region of a stage 37 specimen. Scale bar: 13 μm. (D) Attenuated squamous epithelium covers a large blood vessel between lobes in the folded mesometrial region in a stage 37 specimen. Scale bar: 13 μm. (E) Small blood vessels lie directly beneath columnar epithelium in the folded mesometrial region of the uterus at stage 39. Scale bar = 63 μm. (F) The chorioallantoic membrane opposite the folded mesometrial region exhibits squamous ectodermal cells. Scale bar = 63 μm. AC, attenuated cell; BV, blood vessel; E, uterine epithelium; EC, ectodermal chorion; G, uterine gland; M, muscle.

Figure 4

Immunofluorescent staining (red) with von Willebrand factor to localize blood vessels in the uterus of pregnant Eulamprus quoyii. Nuclei are stained with DAPI (blue). (A) Many small blood vessels form an almost continuous line of staining beneath the uterine epithelium near the mesometrial pole in the uterus of a female with newly ovulated eggs (stages 0–25). Scale bar: 80 μm. (B) Fewer, larger blood vessels in the abembryonic region of the uterus from a newly ovulated female produce more discrete patches of staining. Scale bar: 40 μm. (C) Small sub-epithelial blood vessels in the mesometrial pole of the uterus in a stage 38 specimen. Scale bar: 40 μm. (D) Larger, sparsely distributed blood vessels in the abembryonic region of the uterus in a stage 38 specimen. Scale bar: 40 μm. (E) Negative control omitting the primary antibody shows no staining in a stage 30 specimen. Scale bar: 80 μm. (F) Non-immune control using rabbit IgG instead of the primary antibody shows light, non-specific staining across the whole uterus in a stage 26 specimen. Scale bar: 40 μm. E, uterine epithelium; M, muscle.

Figure 5

A schematic representation of the presence of alkaline phosphatase activity in the uterine epithelium (black bar), uterine blood vessels (dark grey bar) and uterine glands (light grey bar) in Eulamprus quoyii during pregnancy. A solid bar indicates that alkaline phosphatase (AP) activity is present in a particular tissue during the embryonic stage indicated on the horizontal scale. A broken bar indicates that AP activity is present in some samples at this embryonic stage and not others. No bar indicates that AP activity is absent in a particular tissue.

Figure 6

Enzyme histochemical staining to localize alkaline phosphatase activity in the uterus of pregnant Eulamprus quoyii. (A) Alkaline phosphatase activity is visible in protrusions of the uterine epithelium and the underlying blood vessels in the mesometrial region of the uterus in a newly ovulated female (stages 0–25). Scale bar: 13 μm. (B) Alkaline phosphatase activity is present in the uterine epithelial cells and underlying blood vessels in the abembryonic region of the uterus in a newly ovulated female. Scale bar: 13 μm. (C) Alkaline phosphatase activity localizes to the cilia of uterine epithelial cells and underlying blood vessels in the mesometrial region of the uterus in a newly ovulated female. Scale bar: 13 μm. (D) Alkaline phosphatase activity is present in the uterine blood vessels but absent from the uterine epithelium in the mesometrial region of the uterus in a stage 28 specimen. Scale bar: 20 μm. (E) Alkaline phosphatase activity is present in both the uterine glands and blood vessels in the lateral region of the uterus in a stage 34 specimen. Scale bar: 20 μm. (F,G) Alkaline phosphatase activity is present in the uterine glands but absent from the uterine blood vessels and uterine epithelium in the mesometrial region of the uterus in a stage 40 specimen. (H) A negative no-substrate control shows no brown staining in the mesometrial region of the uterus in a stage 25 specimen. Scale bar: 20 μm. BV, blood vessel; Ci, cilia; E, uterine epithelium; G, uterine gland; M, muscle.