Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

Abstract

Background: The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep.

Methods: Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries).

Results: Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries.

Conclusion: These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.

Figure 1

Representative micrograph of ovaries collected from autotransplanted (top ovary) and control (bottom ovary) ewes. Stars (*) indicate large follicles.

Figure 2

Image of oocytes collected from an autotransplanted ewe before (A) and after (B) maturation. Magnification 100×.

Figure 3

Concentrations (mean ± SEM) of FSH (mIU/ml), LH (mIU/ml) and E2 (pg/ml) in serum of control (n = 4; black bars), and autotransplanted (n = 2; open bars) ewes with partially restored ovarian function before FSH-treatment, and on days 15, 16 and 17 after removal of Chronogest sponges. Collection 1 was at the time of autotransplantation, collections 2 and 3 were approximately every 4 weeks within the first two months after autotransplantation, collection 4 was at Chronogest sponge insertion, collection 5 was at sponge withdrawal, and remaining collections were on days 15, 16 and 17 after sponge withdrawal, which corresponds to days 13–15 of the estrous cycle in control ewes.

Figure 4

Representative micrograph of ovarian sections from autotransplanted (A, B, C; left column) and control (D, E, F; right column) ewes stained with hematoxylin and periodic acid Schiff's reagent. Note presence of primordial, primary and secondary follicles (arrows, A and D), early antral follicles (B and E), and preovulatory follicle (C and F). G = granulosa and T = theca in preovulatory follicles in C and F. Bar = 50 μm for A, B, D, E and 100 μm for C and F.

Figure 5

Representative micrograph of staining for factor VIII (black color) in ovarian tissues from autotransplanted (A, B, C, D; left column) and control (E, F, G, H; right column) ewes. Note the presence of factor VIII in blood vessels in the area containing primordial/primary (A, C) and antral follicles (B, F), in the theca layer of preovulatory follicles (C, G), and in capillaries of the CL (D, H). Arrows indicate primordial/primary follicles. G = granulosa and T = theca in preovulatory follicles in B, C, F and G. Control sections did not exhibit any positive staining (see Fig. 8 insert). Bar = 50 μm for A, C, D, E, G, H and 100 μm for B and F.

Figure 6

Representative micrograph of staining for VEGF (black color) in ovarian tissues from autotransplanted (A, B, C, D; left column) and control (E, F, G, H; right column) ewes. Note presence of VEGF in blood vessels in the area containing primordial/primary and antral follicles (A, B, E, F), in the theca layer of preovulatory follicles (C, G), and in the large blood vessels in the ovarian stroma (D, H). Arrows indicate primordial/primary follicles. Control sections did not exhibit any positive staining (see Fig. 8 insert). Bar = 50 μm for A, B, D, E, G, H and 100 μm for B and F.

Figure 7

Representative micrograph of staining for SMCA (black color) in ovarian tissues from autotransplanted (A, B, C; left column) and control (D, E, F; right column) ewes. Note presence of SMCA in blood vessels and connective tissue in the area containing primordial/primary/secondary follicles (A, D), in the thecal layer of preovulatory follicles (B, E), and in larger and smaller blood vessels of ovarian stroma (C, F). Arrows indicate primordial/primary/secondary follicles. G = granulosa in preovulatory follicles in B and E. Control sections did not exhibit any positive staining (see Fig. 8 insert). Bar = 50 µm for A, B, D, G and 100 µm for C and F.

Figure 8

Representative micrograph of staining for PCNA (black color; A-D) and apoptotic cells (brownish color; E-H) in ovarian tissues from autotransplanted (A, B, E, G; left column) and control (C, D, F, H; right column) ewes. Note the presence of PCNA in primordial/primary (arrows; A, C) and preovulatory follicles (B, D), and apoptotic cells in granulosa layer of preovulatory follicle (E, F) an in the CL (G, H). G = granulosa and T = theca in preovulatory follicles in B, D, E and F. Control sections did not exhibit any positive staining (insert). Bar = 50 μm.