Xenotransplantation by injection of a suspension of isolated preantral ovarian follicles and stroma cells under the kidney capsule of nude mice.

OBJECTIVE: To develop and test a novel approach to xenotransplantation of isolated preantral follicles underneath the kidney capsule of immunodeficient mice. DESIGN: Prospective experimental animal study. SETTING: Academic research unit. ANIMAL(S): Healthy adult nude mice. INTERVENTION(S): Bovine ovaries from fetuses (n = 3) and calves (n = 3) were enzymatically disaggregated and subsequently filtered. Isolated preantral follicles were suspended in phosphate buffered saline, and granulosa and stroma cells originating from the ovarian digest served as embedding matrix. The suspension was injected under the kidney capsule of adult nude mice. MAIN OUTCOME MEASURE(S): Fourteen days after transplantation, follicular survival and proliferation in grafts was assessed by histology and proliferating cell nuclear antigen (PCNA) immunostaining, and was compared with ungrafted control tissue. RESULT(S): Primordial follicles decreased from 58.2% in control tissue to 17.1% in transplants in the fetal group, and from 76.0% to 17.2% in the calf group. Concomitantly, primary follicles increased from 13.4% to 62.2% in the fetal group, and from 5.4% to 63.5% in the calf group. Follicular proliferation measured by PCNA immunolabeling exhibited an increase from 40.6% growing follicles to 81.9% in the fetal group, and from 21.0% to 80.7% in the calf group. CONCLUSION(S): The massive follicular activation following transplantation indicates that isolated preantral follicles are able to survive and grow 14 days after renal subcapsular xenotransplantation.

Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing.

This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n=16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and primary follicles significantly increased. The proportion of secondary and antral follicles after SRF was significantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles.

Development and practical applications of a method for repeated transvaginal, ultrasound-guided biopsy collection of the bovine ovary.

In response to the increasing research into primordial and preantral follicular dynamics, a device for transvaginal, ultrasound-guided biopsy collection of the bovine ovary was developed and tested. The new device is based upon a commercially available Ovum Pick-up instrument and consists of a modified needle guidance system, which has been equipped with a trocar needle and caries a 60 cm long true-cut biopsy needle. Biopsies are captured in a 20mm long and 2mm wide specimen notch. In the present experiment, 10 cows were subjected to a twice weekly biopsy regime over a four-week period. A total of 208 attempts at biopsy collection were made, and 141 tissue samples collected (success rate of 68%). Through histological and immunological analyses, these tissue samples have been shown to contain primordial and preantral follicles. At the end of the trial period, several of the donor cows were slaughtered at timed intervals, and the ovaries were harvested for assessment of the damage inflicted by the repeated biopsy procedure. Post mortem ovaries were inspected macroscopically and examined by conventional histological staining. In ovaries retrieved 2 days after the last biopsy session, blood clots were macroscopically apparent throughout the ovaries. Histological examination showed increased infiltration of red blood cells in the ovarian stroma. Analysis from ovaries collected at subsequent slaughter points revealed reduced infiltration of blood, and clear indications of resumed antral follicle development were apparent towards the end of the first month after the trial period. We conclude that the biopsy sampling technique is a repeatable procedure which could serve as a renewable source of primordial and preantral follicles for culture, and as an in vitro model for the study of preantral follicular dynamics.