Understanding the Inguinal Sinus in Sheep (Ovis aries)-Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors

Abstract

Post-parturient behavior of mammalian females is essential for early parent-offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

Keywords: ESR1; ESR2; PGR; PRLR; chemical compounds; inguinal sinus; morphology; transcription; triterpenoids.

Figure 1

By abduction of the hind-left leg one can observe the skin pouch (IGS) over the mammary gland. On the right, the arrow points out the IGS and its yellowish secretion.

Figure 2

Histology-Bar = 100 µm. Van Gieson’s Stain. (A) Organization of the inguinal sinus presenting sebaceous (black arrows) and acinar glandular fields (white arrow); (B) collagen fibers sustain the secretory epithelium of the acinar glands that presents; (C) cellular protrusions towards the glandular lumen (apocrine secretion). Parenchyma rich in glycogen can be observed and its amount varies in different areas of the gland being the secretory cells in different stages of secretion production in different areas of the gland–PAS; (D–F); In (E) the arrow points out a myoepithelial cell. (D) = 40× magnification; (E) = 1000×. Visualizations of lipid granules with Sudan black stain; (G) Alcian blue stain depicts mucin content; (H,I) Magnification = 1000×.

Figure 3

Scanning electronic images of IGS. In (A) (bar = 500 µm); (B) (bar = 50 µm) and (C) (bar = 10 µm) it is clear that apocrine glandular structures appear in clusters inside the IGS. Luminal surface can show a paved appearance; (D) bar = 10 µm or an irregular one resulting from the secretory process. Secretory cells appear in different stages of differentiation, where fragments of secretion are being “pinched off” exhibiting secretory vesicles (secretion blebs), while cells maintain a clear demarcation with neighboring cells by means of rows of microvilli; (D) (bar = 10 µm); (E) (bar = 5 µm) and (F) (bar = 5 µm). A progressive gland filling process results on the upsurge of bulge-like structure; (G) (bar = 5 µm) and (H) (bar = 5 µm). Some cells appear to be in a transitional process where demarcation with surrounding cells is no longer as visible resulting from the development of apical protrusions that in a final stage denote a smooth plasma membrane devoid of microvilli and covering the protrusions; (I) bar = 10 µm.

Figure 4

Qualitative PCR electrophoresis gel and dissociation curves of real-time PCR confirming estrogen receptor 1 (ESR1), and 2 ESR2, progesterone receptor (PGR) and prolactin receptor (PRLR) gene transcription in the IGS in different phase of the estrous cycle. Green arrow indicates the specific gene band. (F) follicular phase; (ML) mid luteal phase; (M) DNA marker; bp (base pairs). All primers validated for 80 nM in the real time PCR run. Single product confirmation with the single peak in the dissociation curve.

Figure 5

Examples of laser-scanning confocal fluorescence (LSC—lens 63.03 oil) images of IGS. One can observe immunoreactivity towards ESR1, ESR2, PGR and PRLR in cells of the apocrine glands labeled with PE and stained for the different receptors (fluorescence in green). Use of To-Pro-3 iodide for nuclear counterstaining (fluorescence in red).

Figure 6

Flow cytometry analysis of gated cells of IGS. Examples of gated and dot plots and histograms showing the expression of ESR1, ESR2, PLRL and PGR. Shown flow cytometry data depict a positive cell expression towards the receptors under evaluation.

Figure 7

Chromatogram analysis revealed the presence of triterpenoids as marker chemical classes of post-partum ewes (PP), pregnant ewes (P) and non-pregnant (NP) samples, and a chemical profile that clearly portrait three major bands: 1, 2 and 3. These compounds were absent on S, the surrounding skin sample used as a negative control. The background color of the chromatogram is not uniform, which is a normal and recognized situation and does not interfere with the interpretation of the results.