Meloxicam and Buprenorphine Treatment after Ovarian Transplantation Does Not Affect Estrous Cyclicity and Follicular Integrity in Aged CBA/J Mice

Angiogenesis, the formation of new blood vessels, is important for the survival of ovarian transplants and the restoration of ovarian functions. Without angiogenesis, transplanted ovarian tissue becomes more susceptible to tissue damage and necrosis. Administration of analgesics for pain management has been shown to decrease angiogenesis, which can influence transplant success especially in aged animals. Aging and the effects of hypoxia after transplantation decrease reproductive viability of the ovarian transplant; therefore, it is important to understand the additional effects of analgesics on aged animal models. The present study investigated the effects of two analgesics, buprenorphine, an opiate, and meloxicam, a non-steroidal anti-inflammatory drug (NSAID), on the reproductive indicators related to estrous cyclicity and follicular integrity after ovarian transplantation of young ovaries into aged CBA/J mice. These aged females did not show any different reproductive responses when treated with either buprenorphine or meloxicam. No significant differences were observed in estrous cycle length, the onset of estrous cycling, the regularity of estrous cycles, and the proportion of viable follicles and total number of follicles per ovarian sample across treatment groups.     

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.     

Vascular remodeling and angiogenesis in ectopic ovarian transplants: a crucial role of pericytes and vascular smooth muscle cells in maintenance of ovarian grafts

Cancer patients, treated by either chemo- or radiotherapy, frequently suffer from ovarian failure and infertility. One of the new emerging techniques to preserve reproductive potential of such patients is cryopreservation of ovarian fragments prior to treatment and their retransplantation after healing. A major obstacle in survival of the ovarian implants is vascular failure, which leads to tissue necrosis. In order to investigate the role of angiogenesis in implant preservation, we used a xenograft model in which rat ovaries were transplanted into immunodeficient mice. Graft reception and maintenance were monitored by magnetic resonance imaging (MRI) and histology. Two transplantation sites were explored, i.e., subcutaneous and intramuscular. Comparison between these two transplantation sites revealed the importance of vascular smooth muscle cells and pericytes in sustaining vascular and tissue integrity. Histological examination of the grafts, at different time points and sizes, revealed that loss of perivascular cells preceded damage to endothelial cells and was closely correlated with loss of follicular and oocyte integrity. Intramuscular implantation provided better maintenance of implant perivascular cells relative to subcutaneous implantation. Accordingly, follicular integrity was superior in the intramuscular implants and the number of damaged follicles was significantly lower compared with the subcutaneous transplantation site. These results suggest that improving ovarian implant maintenance should be directed toward preservation of perivascular support.     

Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals

Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

Developmental Status: Impact of Short-Term Ischemia on Follicular Survival of Whole Ovarian Transplantation in a Rabbit Model

Ischemia is the first mechanism that provokes the loss of follicles in ovarian grafts over the long term. In whole ovarian transplantation, it remains unknown, however, how changes in follicular development are influenced by short-term ischemia. Fresh whole ovarian orthotopic auto-transplantation was performed in rabbits with 45 min ischemia, and the impact of ischemia on follicular survival and development status was evaluated at different time-points (1 day, 3 days, 1 week, 2 weeks and 1 month). Assessment of follicular quantity and morphology was carried out via histologic analysis. Follicle proliferating status was evidenced by immunostaining with proliferating cell nuclear antigen (PCNA), and the Hedgehog signaling pathway (Patched and Gli); was verified via TUNEL assay. Quantitative PCR was carried out to quantify the mRNA of target genes including PCNA, Patched, Gli, Caspase 3, Bax, and Bcl-2. Compared with its contralateral fresh controls, the morphology, proliferation and apoptosis of the follicles in the grafts showed no significant differences and most primordial follicles were quiescent. However, morphology and proliferation status were significantly decreased 1 week after grafting, in comparison with the longitudinal grafting time. Patched and Gli in the Hedgehog signaling pathway were activated in only the follicles of the grafts. Short-term ischemia slightly impacts follicular survival and development status in whole ovarian grafting. Receiving intervention in the first week post-transplantation might be helpful.     

Histological evaluation of the effect of VEGF on auto-transplanted mouse ovaries

One of the most important factors affecting survival rate of ovarian follicles during transplantation period is proper vascular development. The objective of the study was to evaluate the effect of vascular endothelial growth factor (VEGF) on auto-transplanted ovarian tissue. Twenty-one-day-old female mice (n?=?30) transplanted as control group and 21-day-old female mice (n?=?40) were divided into 4 groups that were treated with 0.5, 1, 2 and 4?µg/mL of VEGF directly injected to auto-transplanted ovarian tissue. Twenty-one days after transplantation, mice were treated with 7.5 IU pregnant mare serum gonadotropin and human chorionic gonadotropin. Transplanted ovaries were removed and sections were prepared from transplanted tissues for staining. The most effective dosage of VEGF on transplanted tissue was determined over H&;E (hematoxylin and eosin) staining results. Slides were compared using TUNEL staining and CD31 assay for the most effective dosage. The percentages of preantral and antral follicles were not significantly different between transplanted group with 4?µg/mL VEGF and non-transplanted group. Lower apoptotic areas and higher CD31 expression were observed in transplanted ovaries treated with 4?µg/mL VEGF when compared to transplanted ovaries without VEGF treatment. VEGF positively affects the quality of ovarian tissue during transplantation. Survival rate of follicles and follicular development has improved with the effect of VEGF.     

Follicular development in cryopreserved Common Wombat ovarian tissue xenografted to Nude rats

The Northern Hairy-nosed Wombat (Lasiorhinus krefftii) is a highly endangered marsupial species and every possible option for sustaining the species needs to be explored. One important approach may be the development of assisted reproductive technologies in the non-endangered Common Wombat (Vombatus ursinus) and Southern Hairy-nosed Wombat (Lasiorhinus latifrons) for application in breeding the Northern Hairy-nosed Wombat.In this study, it was examined whether cryopreserved Wombat ovarian tissue would develop following xenografting to immunologically deficient rats. Ovarian tissue was collected from Common Wombats (n = 3) and cryopreserved as small cortical pieces. After thawing the cortical pieces were grafted underneath the kidney capsule of Nude rats (n = 16). The grafts were recovered at 2, 4, and 10 weeks after transplantation and their gross and histological appearance investigated. Two weeks after grafting (n = 2), the tissue was revascularized and healthy primordial follicles were present. At week 4 (n = 2), some follicular development was present. At week 10, six rats received human chorionic gonadotrophin (hCG) to trigger follicle and oocyte maturation while another six rats were not given any treatment. The administration of hCG did not induce preovulatory follicles and oocyte maturation although type 5 follicles were present in ovarian tissue collected 10 weeks posttransplantation in both treated and untreated groups. This study demonstrates for the first time that Wombat ovarian tissue can survive and function when grafted into immunocompromized rats and that Wombat ovarian follicles can be recruited to growth and development in an ovarian xenograft. This model system has the potential to produce mature oocytes from endangered species for use in assisted reproductive technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and mature oocytes from non-endangered species for nuclear transfer which may be necessary for the preservation of critically endangered species.     

丹参对冻融入胎儿卵巢组织异种移植早期血管生成的影响

目的：研究冻融人胎儿卵巢组织移植早期血管生成过程中微血管形态和密度的改变以及血管生成相关基因mRNA的表达;探讨丹参注射液对移植物血管生成的影响。方法：冻融胎儿卵巢组织异种移植至裸鼠肾被膜下,按给药不同分为对照组（生理盐水）和丹参组（丹参注射液每只0．09g／d）,分别于移植后48h、7d和28d回收移植物。结果：移植后两组卵巢组织微血管密度均明显增多;对照组移植后7d血管密度达峰,丹参组血管密度在移植后48h即已显著上升,此后两个时间段保持相对平稳。Angiopoietin-2mRNA表达在移植后48h两组均显著升高,丹参组上升幅度大于对照组（P〈0．05）。结论：冻融人胎儿卵巢组织新血管生成开始于异种移植后48h内,移植后7d组织内微血管密度达峰。丹参在移植早期应用可以促进移植后血管生成,其机制可能与它增加了血管生成相关因子Ang-2mRNA的表达有关。     

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.     

The effect of pinealectomy, melatonin and leptin hormones on ovarian follicular development in female Syrian hamsters (Mesocricetus auratus)

We studied the effects of melatonin and leptin hormones on ovarian follicular development in intact and pinealectomized female Syrian hamsters. We first monitored the oestrous cycle of the hamsters by the vaginal smear samples throughout a ten day period to start the injections simultaneously in all groups and performed saline, melatonin and leptin hormone injection groups for both control and pinealectomized hamsters. Then the injections were applied for four days starting the oestrus phase of the cycle and the ovaries were removed for preparation of histological analysis. We measured the diameters and the numbers of the follicles and we classified the follicles according to the number of the granulosa cell layer. Leptin hormone injection increased melatonin hormone injection decreased the number and the diameter of the follicles. The stimulating effect of the leptin hormone was more pronounced in the pinealectomized group. The results of the present study indicate that the removal of the pineal gland and leptin hormone administration are playing a stimulatory while melatonin hormone administration is playing an inhibitory role on the follicular development in female Syrian hamsters.     

人胎儿卵巢异种移植的组织学研究

目的探讨人胎儿卵巢组织异体移植的最佳条件。方法观察胎儿卵巢进行异种移植后的组织学变化,并计算各组移植卵巢内各期卵泡的发育情况及卵泡构成比。结果新鲜的卵巢组织、体外培养的卵巢组织以及冷冻复苏后经体外培养的卵巢组织移植后,其内有原始卵泡发育,而且可见丰富的血管网;未见明显的淋巴细胞浸润现象。结论人胎儿卵巢组织(包括新鲜的卵巢组织、体外培养的卵巢组织以及冷冻复苏后经体外培养的卵巢组织)异种移植后原始卵泡在受体体内能进一步发育,生长卵泡分泌的雌激素有调节子宫内膜周期性变化的作用。     

Follicular microvasculature in the porcine ovary

The microvasculature of porcine ovaries, with special regard to the follicles in the interstitial-stromal tissue, was studied by scanning electron microscopy (SEM) of vascular corrosion casts. Porcine ovaries displayed several coiled arteries in the hilus and many branches with small diameters and a tightly spiraling configuration in the cortical areas. However, small arterioles became straight before entering vascular complexes of follicles and finally divided into capillaries. Vascular baskets of various sizes (150-9,900 micro m in diameter) and architecture related to follicles in various developmental stages were observed in the ovarian cortex. Small follicles (150-300 micro m in diameter) began with a polygonal meshwork of a few large capillary meshes and developed to an obvious spherical microvascular network with a thin single layer of capillaries when reaching 500-700 micro m in diameter. The microvascular architecture of follicles 1,000-2,000 micro m in diameter developed further and had a three-layer vascular plexus. With a diameter of more than 2,000 micro m, the microvasculature of antral follicles was arranged as an inner vascular plexus of about 25 micro m, a middle plexus of about 100 micro m, and an outer capillary plexus of about 30 micro m in thickness. The present observations indicate that follicular vascular baskets of diverse sizes and architecture in various developmental stages support the gradual increase of follicular blood flow during follicle growth in the pig.     

Effects of in vitro culture of mouse fetal gonads on subsequent ovarian development in vivo and oocyte maturation in vitro

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 µm in diameter and had competence to resume meiosis in vitro . When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences ( P  < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.     

Separation of native and latent forms of human antithrombin by hydrophobic interaction high-performance liquid chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumor growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0.     

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.     

Expression of the human fast-twitch skeletal muscle troponin I cDNA in a human ovarian carcinoma sup-presses tumor growth

To explore the efficiency and mechanism of ovarian carcinoma gene therapy with the human fast-twitch skeletal muscle troponin I gene (TnI-fast), TnI-fast cDNA was transferred into human ovarian adeno-carcinoma cell-line SK-OV-3. In vitro, the cell growth and cell cycle of TnI-fast-, vector-, and mock-transfected cells were determined by MTT and flow cytometry assay, respectively. The condi-tioned media of TnI-fast-, vector-, and mock-transfected SK-OV-3 cells were collected, and the cell pro-liferation inhibiting rates of human umbilical cord venous endothelial cells (HUVECs) by the three conditioned media were assayed. All the three cell lines were implanted into node mice, and the tumor growth, cell apoptosis, angiogenesis, and expression of TnI-fast were observed or analyzed, respec-tively. In vitro, expression of TnI-fast protein had no inhibiting effect on the growth of the dominant and stable transfectant cells, but endothelium, when compared with vector-transfected cells and nontrans-fected parental SK-OV-3 cells. Implantation of stable clone expressing TnI-fast in the female BALB/c nude mice inhibits primary tumor growth by an average of 73%. The nude mice grafts expressing TnI-fast exhibit a significant decrease of microvascular density, a higher rate of tumor cells apoptosis and a comparable proliferation rate as control. Our study, to our knowledge, shows the slowed down growth of the primary ovarian carcinoma, suggested that grafts were self-inhibitory by halting angio-genesis. Our data might also provide a novel useful strategy for cancer therapy by antiangiogenic gene therapy with a specific angiogenesis inhibitor TnI-fast.     

Preparative conversion of native human antithrombin to the latent form

Human native antithrombin (AT) can be converted to a partially denaturated form of AT, known as latent AT (L-AT). This latent form of AT has been shown to exhibit strong antiangiogenic activity and also to suppress tumor growth in mice models. In the present work, a method is presented which induces the conversion of native AT to L-AT, using incubation at 60 degrees C, for 16 h, with 0.9 M ammonium sulfate, in 5mM Hepes buffer, pH 7.4, giving a recovery of more than 70%. L-AT was determined by integration of the low heparin affinity peak when analyzed by the affinity chromatography method. Native polyacrylamide gel electrophoresis was used to show that the preparation contained no aggregates. Hydrophobic interaction chromatography was also used for the separation of AT and L-AT.