Donor-host involvement in immature rat testis xenografting into nude mouse hosts

Abstract

Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted immature donor tissue and 2) the recipient's cells and tissues contribute to the functional recovery of a "testicular xenograft." We address whether recipient cells migrate into the testicular parenchyma and whether the circulatory connection between the donor testicular tissue and the recipient is established by ingrowing host or outgrowing donor blood vessels. Although this issue has been repeatedly discussed in previous xenografting studies, so far it has not been possible to unequivocally distinguish between donor and recipient tissues and thus to identify the mechanisms by which the circulatory connection is established. To facilitate the distinction of donor and recipient tissues, herein we used immature green fluorescent protein-positive rat testes as donor tissues and adult nude mice as graft recipients. At the time of graft recovery, donor tissues could be easily identified by the GFP expression in these tissues, allowing us to distinguish donor- and recipient-derived blood vessels. We conclude that the circulatory connection between graft and host is established by a combination of outgrowing small capillaries from the donor tissue and formation of larger vessels by the host, which connect the graft to subcutaneous blood vessels.

PLATE I. Figures 1 and 2.. FIG. 1. Schema demonstrating the grafting procedure. GFP-positive rat pups can be distinguished from GFP-negative littermates by UV illumination (left). GFP-positive testes are excised (I), transversely cut in halves (II), and grafted under the dorsal skin using G13 transfer needles (III).FIG. 2. In vivo fluorescence reflectance image of a nude mouse carrying GFP-positive rat testicular grafts imaged at 3–10 days after graft implantation. The grafted GFP-positive tissues can be clearly detected under the dorsal skin of the host (arrowheads). Note the increasing size of the grafts over time.

FIG. 3.

Images of xenografts derived from immature GFP-positive rat testes showing the grafts still attached to the partly removed dorsal skin of the recipient. a) Two grafts (arrowheads) show small blood vessels in capsule and parenchyma. A subcutaneous blood vessel (scv) can be seen directly connected (arrow) to a vessel supplying one of the grafts (gsv). b) Three grafts (asterisk) show fine blood vessels. Note the bigger subcutaneous vessels (arrowhead). c and d) GFP-positive grafts (arrowheads) can be seen lying on the GFP-negative background of the dorsal skin of the hosts. Blood vessels are clearly blood filled (arrows). The large graft-supplying blood vessel (d, arrow) appears GFP negative and is thus most likely of host origin.

FIG. 4.

Fluorescent micrographs showing cryosections of GFP rat testis tissue grafts retrieved at 4 wk after implantation. GFP (green) is visualized by native fluorescence, and SMA (red) is visualized by immunohistochemistry. Nuclei are labeled with DAPI (blue). a) Note the GFP-positive inner capsule (arrow), the GFP-negative outer capsule (arrowhead), the blood vessels in the interstitium (double arrow), and the seminiferous tubules with ongoing spermatogenesis and lumen (asterisk). b) Note the GFP-positive (arrow) and GFP-negative (arrowhead) blood vessels in the GFP-negative part of the capsule. c) A GFP-positive blood vessel can been seen protruding into the GFP-negative part of the capsule (arrow).

FIG. 5.

Fluorescent micrographs showing paraffin sections of GFP rat testis tissue at 8 wk (a–c) and 4 wk (d) after implantation. GFP (green) and SMA (red) are visualized by immunohistochemistry; nuclei are labeled with DAPI (blue). a) Seminiferous tubules are surrounded by an SMA-positive basement membrane (arrow). Note the GFP-positive interstitium (asterisk). b) Few GFP-positive cells can be detected in the otherwise GFP-negative part of the capsule (arrows). c) Numerous small blood vessels are present in the interstitium (arrows). The inset in c (same magnification as panel c) shows a technical control for the immunohistochemical staining (omission of the primary antibodies). d) A GFP-positive blood vessel can be observed within subcutaneous muscles of the recipient mouse (arrow). Nonnucleated erythrocytes (cells appearing green with no nuclear DAPI label within the vessel lumen) can be detected within this blood vessel, underlining the functionality of this particular vessel.

FIG. 6.

Fluorescent micrographs showing paraffin sections of GFP rat cell grafts at 4 wk after implantation. GFP (green) and SMA (red) are visualized by immunofluorescence. Nuclei are labeled with DAPI (blue). The bar shown in c also applies to a and b. a) The graft is surrounded by a GFP-positive (arrow) and GFP-negative (arrowhead) capsule. Note the GFP-positive and GFP-negative cells in the interstitium (asterisk) and the poorly differentiated seminiferous tubules. b) Some grafts show a limited number of GFP-positive cells in the interstitium (asterisk), and only cord-like structures and tubular shadows can be observed. In some grafts, no seminiferous cords or tubules can be observed, although a few GFP-positive cells and tissues can still be found in the tissues (arrows). c) Few GFP-positive tubules can be observed within this graft (arrows), between a series of tubular shadows.