Fig 1.
Illustration of experimental procedures performed to assess the effects of cooling and VEGF on heterotopic ovarian tissue autotransplantation in mares.
(A) Collection of ovarian fragments by ultrasound-guided transvaginal biopsy pick-up method; biopsy needle notch without and with a harvested ovarian fragment; fragments with surgical thread ready to be transplanted; and two transplanted fragments. (B) Experimental design, treatments, and end points. (C–H) Representative images of ovarian grafts (white dashed circles) during the harvesting process at day 7. (C–E) and (F–H) Images obtained from two different animals. (E and H) Magnified images of recovered grafts. (F) Ovarian fragment embedded and covered with subcutaneous tissue. (C, D, G) Exposed grafts at the surgical site. (C, D, F, G) Bars = 3 mm, and (E, H) bars = 1 mm. (I–L) Representative images of morphologically (I–K) normal and (L) abnormal preantral follicles in grafted ovarian tissue previously cooled. (I, L) Primordial, (J) primary, and (K) secondary follicles. Magnification = 400×; bars = 20 μm.
Fig 2.
(A) Schematic design of the transplant sites with the different regions (R1, R2, R3, R4, R5, and R6) used to evaluate the local blood flow in each neck side [i.e., left side = non-cooled (25°C) transplant treatments, and right side = cooled (4°C) transplant treatments]. Implanted ovarian tissues, associated or not to previous exposure to VEGF, are shown in red. (B–C) Mean (± SEM; n = 8 animals) blood flow area comparison between non-cooled and cooled fragments (VEGF–/+ combined treatments) at (B) the different regions of the transplant site in each neck side, and (C) different days post-grafting. a,bWithin each region, different letters indicate differences between non-cooled and cooled treatments. *Indicates the first significant increase or decrease for each treatment, and a difference between treatments within a day. The probabilities for a treatment effect, day effect, and treatment-by-day interaction are shown. (D–K) Representative color-Doppler ultrasonograms indicating the presence of different degrees of blood perfusion ("hyperemia") in a specific region of the transplant site at days 2, 4, 6, and 7 post-grafting using (D, F, H, J) non-cooled and (E, G, I, K) cooled ovarian tissues. Color signals indicate blood flow in each ultrasonogram.
Fig 3.
Mean (± SEM; n = 8 animals) percentages of (A–E) morphologically normal preantral follicles (i.e., primordial to secondary-stage follicles) and (F–J) developing follicles (i.e., normal developing follicles divided by the total number of normal preantral follicles multiplied by 100) after 7 days of heterotopic ovarian tissue autotransplantation in mares.
(A and F) Comparisons of Fresh control with other treatments (one-way ANOVA) and (A–J) between non-transplant and transplant treatments (two-way ANOVA) were made. The effects of (B and G) procedure (non-transplant vs. transplant) using cooled tissue, (C and H) cooling/temperature (transplants at 4°C vs. 25°C) regardless of VEGF, (D and I) VEGF on transplant regardless of temperature, and (E and J) VEGF on cooled ovarian tissue were evaluated after grafting. *Indicates a difference (P < 0.05) between the Fresh control and another group. a,bWithin any effect analyzed, different letters indicate differences between treatments. P-values for the main analyses are shown.
Fig 4.
Mean (± SEM; n = 8 animals) percentage of (A–E) follicular density and (F–J) stromal cell density after 7 days of heterotopic ovarian tissue autotransplantation in mares.
(A and F) Comparisons of Fresh control with other treatments (one-way ANOVA) and (A–J) between non-transplant and transplant treatments (two-way ANOVA) were made. The effects of (B and G) procedure (non-transplant vs. transplant) using cooled tissue, (C and H) cooling/temperature (transplants at 4°C vs. 25°C) regardless of VEGF, (D and I) VEGF on transplant regardless of temperature, and (E and J) VEGF on cooled ovarian tissue were evaluated after grafting. *Indicates a difference (P < 0.05) between the Fresh control and another group. a,bWithin any effect analyzed, different letters indicate differences between treatments. P-values for the main analyses are shown.
Fig 5.
Mean (± SEM; n = 8 animals) (A–C) new vessel density, (E–G) percentage of stromal cells labeled with caspase 3, and (I–K) percentage of nucleolus organizer regions (NORs) per nucleus of stromal cells after 7 days of heterotopic ovarian tissue autotransplantation in mares. (A, E, I) Comparisons of Fresh control with other treatments (one-way ANOVA) and between transplant treatments within different conditions (two-way ANOVA) were made. The effects of (B, F, J) cooling/temperature (transplants at 4°C vs. 25°C) regardless of VEGF, and (C, G, K) VEGF on transplant regardless of temperature were evaluated after grafting. *Indicates a difference (P < 0.05) between the Fresh control and another group. a,bWithin any effect analyzed, different letters indicate differences between treatments. P-values for the main analyses are shown. Representative histological images with (D) the presence of CD31-stained new vessels, (H) stromal cells labeled with caspase 3, and (L) NORs in stromal cell nucleus are shown (arrowheads) for the Fresh control and transplant treatments. (D and H) Magnification = 400×, scale bars = 20 μm; and (L) Magnification = 1000×, scale bar = 10 μm.
Fig 6.
Mean (± SEM; n = 8 animals) collagen type (A–C) I and (D–F) III fibers after 7 days of heterotopic ovarian tissue autotransplantation in mares. (A and D) Comparisons of Fresh control with other treatments (one-way ANOVA) and between transplant treatments within different conditions (two-way ANOVA) were made. The effects of (B and E) cooling/temperature (transplants at 4°C vs. 25°C) regardless of VEGF, and (C and F) VEGF on transplant regardless of temperature were evaluated after grafting. *Indicates a difference (P < 0.05) between the Fresh control and another group. a,bWithin any effect analyzed, different letters indicate differences between treatments. P-values for the main analyses are shown. Representative histological images of ovarian tissue stained with Picrosirius red stain showing collagen type (G, K, O, S, W) I and (H, L, P, T, X) III fibers, and (I, M, Q, U, Y) merged images are depicted for the Fresh control and transplant treatments. (J, N, R, V, Z) 3D images illustrate the spatial distribution of the two types of collagen in each group. Image magnification = 400×; scale bars = 20 μm.