Figure 1.
Testis lymphangiogenesis is initiated during late gestation in mice, as visualized by confocal microscopy of whole-mount Prox1-EGFP gonad-mesonephros complexes.
A) At 16.5 dpc, an extensive lymphatic network is present in the adjacent mesonephros, but excluded from the testis. B) Lymphatic vessels first appear on the testis surface from 17.5 dpc, seemingly by continuous growth of the vessels already present in the mesonephros. C) At 18.5 dpc, lymphatic vessels continue to sprout across the surface of the testis. D) Prominent Prox1-EGFP positive vessels of the 18.5 dpc testis also express endogenous PROX1, E) and the lymphatic marker NRP2, F) both overlapping with Prox1-EGFP expression. RT = rete testis; scale bars: A = 500 µm, D = 200 µm.
Figure 2.
Lymphatic vessels sprout across, but not beyond, the testis cap at 17.5 dpc.
Representative images captured from optical projection tomograph, also represented in Vid S1. A) During late gestation, EGFP-positive lymphatic vessels are seen growing from pre-existing vessels in the spermatic cord before sprouting across the testis surface. B) The fetal testis also contains a rich network of blood vessels visualised by ENG staining, but C) the EGFP-positive vessels do not overlap with the more extensive blood vasculature. Yellow areas correspond to lymphatic vessels (green) and blood vessels (red) in different planes. D) 3-D model of Prox1-EGFP positive lymphatic network during initial development. E) Magnified region showing two EGFP-positive lymphatic vessels running parallel to the coelomic (arterial) vessel and F) magnified region of the rete testis. T = testis; E1 = head of epididymis; E2 = tail of epididymis; CV = coelomic vessel; RT = rete testis; scale bars: A = 500 µm, B = 250 µm.
Figure 3.
Lymphatic vessels are limited to the tunica albuginea in adult testis.
A–C) EGFP-positive vessels are readily observed across the surface of the adult testis emanating from the spermatic cord (asterisk). Strong EGFP expression is also observed within the seminiferous tubules. D–F) Sectioned Prox1-EGFP transgenic adult testis co-stained with the Leydig cell marker HSD3B1 and counterstained with DAPI revealed no lymphatic vessels inside the testis, but within the tunica albuginea (arrow). Additional EGFP expression was verified within the seminiferous tubules (encircled) and localised to spermatids closer to the lumen. G–I) Sectioned Prox1-EGFP adult epididymis co-stained with the smooth muscle cell marker ACTA2 to demarcate the vas deferens and counterstained with DAPI showed prominent EGFP-positive lymphatic vessels, but also EGFP-positive sperm cells. BV = blood vessel; scale bars C = 600 µm, D = 100 µm, G = 300 µm.
Figure 4.
Lymphatic vessels develop in the postnatal ovary from around 10 dpn. A)
At 7 dpn, EGFP-positive vessels are observed in along one side of the uterine horn and in the ovarian ligaments, but the ovary (encircled) is still devoid of lymphatics. B) At 10 dpn, the ovary possesses a lymphatic network. Lymphatic vessels sprouting laterally at distinct regional distances from a pre-existing vasculature along the length of the uterus have almost encircled the entire uterine horn. C) At 14 dpn, the ovary possesses a distinct lymphatic network and the uterine horn has developed a strikingly segmented lymphatic network encircling the entire tissue D) The adult ovary maintain a high Prox1-RGFP expression and the uterine horn has developed a extensive mesh of lymphatic vessels. E) Prominent Prox1-EGFP positive vessels of the 9 week uterine horn also express endogenous PROX1, and F) the lymphatic marker NRP2, G) both overlapping with Prox1-EGFP expression. OL = ovarian ligament; U = uterine horn; scale bar = 200 µm.
Figure 5.
The adult ovary possesses an extensive lymphatic network. A–C)
In the ovarian cortex, secondary follicles (encircled) with DDX4-positive oocytes are devoid of Prox1-EGFP positive lymphatic vessels, but lymphatic vessels are readily observed in close proximity. D–F) The ovarian medulla visualized by staining of the theca cell marker CYP11A1, contains a rich network of lymphatic vessels. G–I) The granulosa layer of secondary follicles localized by FOXL2-staining is devoid of lymphatic vessels, but with Prox1-EGFP positive vessels localizing very close to the border between the theca and granulosa layers (dotted circle). Oocytes of primary follicles containing a single layer of FOXL2-positive granulosa cells express a discernable level of Prox1-EGFP (arrow). Scale bar A = 100 µm.
Figure 6.
The adult ovary possesses a rich lymphatic network largely overlapping with the blood vasculature.
Representative images captured from optical projection tomograph, also represented in Vid S2. A) The mature ovary contain a dense network of lymphatic vessels that emanate from the rete ovarii, and B) a rich network of ENG-positive blood vessels. C) Lymphatic vessels also expressing LYVE1 are generally localised to the ovarian and extraovarian rete. D) 3-D model of Prox1-EGFP positive lymphatic network as compared to E) LYVE1-positive lymphatic vessels and ENG-positive blood vessels F) Merged image of ENG, EGFP and LYVE1 expression in the adult ovary reveals distinct patterning of the blood and lymphatic network. Oc = ovarian cortex; Om = ovarian medulla; RO = rete ovarii; F = follicle; scale bar = 1 mm.