Antioxidative effect of melatonin on cryopreserved ovarian tissue in mice

Cryopreservation of ovarian tissues (OTs) has become the most effective way to preserve the fertility of female cancer patients. However, cryopreservation of OTs is still relatively at an experimental stage. The aim of study is to examine the effect of melatonin (MTL) on cryopreserved-thawed OTs. Fragments of OTs were cryopreserved in medium containing different concentrations (0 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1 mM) of MLT. The endogenous enzymes (GSH-PX, GSH, SOD, CAT and T-AOC), MDA and ROS levels were all evaluated after cryopreservation. Our results showed that the 0.1 mM of MLT significantly improved the survival and diameter of follicles (P < 0.001). Meanwhile, the antioxidant enzymes activities (including GSH-PX, GSH, SOD, CAT and T-AOC) were enhanced and MDA content were significantly decreased in 0.1 mM of MLT group compared to other groups (P < 0.001). Additionally, compared to the control group, MTL of 0.1 mM resulted in a significantly lower ROS level. In conclusion, MLT protects the quality of cryopreserved OTs by decreasing oxidative stress level and the optimal concentration is 0.1 mM.     

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.     

In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.     

Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at ∼60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 ± 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.     

In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue

Achieving full in vitro growth of oocytes of both domestic animals and humans remains a major challenge. The objective of this study was to examine the in vitro development of primary follicles isolated enzymatically from cryopreserved sheep ovarian tissue. In Experiment 1, isolated primary follicles (mean diameter 60.1+/-0.78microm) were cultured in serum-free medium on fibronectin-coated wells for 42 days. Initially follicular structure was lost as granulosa cells plated down, but by Day 7 two distinct morphologies began to emerge. Nineteen out of 36 oocytes were gradually re-surrounded by granulosa cells, forming follicle-like units (reorganized follicles), and the remaining 17 were not (non-reorganized follicles). On Day 2, there was no difference in diameter of oocytes between reorganized and non-reorganized follicles. The diameter (mean+/-S.E.M.) of oocytes of reorganized follicles increased (P<0.05) from 47.1+/-2.2microm to 65.3+/-2.6microm between Day 2 and Day 42, respectively, but that of oocytes of non-reorganized follicles showed no change. In Experiment 2, oocyte growth and granulosa cell differentiation during long-term culture of primary follicles (>42 days) were examined. Oocytes of reorganized follicles reached a maximum diameter of 75.4+/-2.0microm, a size equivalent to that of oocytes of ovine secondary follicles. Using RT-PCR, mRNA for follicle stimulating hormone receptor was detected in granulosa cells of freshly isolated secondary follicles and of long-term cultured reorganized follicles, but not of non-reorganized follicles. In Experiment 3, we tested if the culture conditions could support further oocyte growth in secondary follicles. The oocytes from enzymatically isolated secondary follicles increased in diameter from 77.7+/-1.6microm to 98.8+/-2.1microm (P<0.05) during 28 days in culture. The changes in oocyte size and in gene expression by granulosa cells support the conclusion that isolated ovine primary follicles developed in vitro to reach the secondary follicle stage.     

Effect of reduced ovarian tissue on cyclicity, basal hormonal levels and follicular development in old rats

Reduction of the number of growing follicles was proposed to contribute to the decline in reproductive performance with aging (Butcher and Page, 1981). To investigate the effects of a reduced number of follicles, rats which maintained regular estrous cycles at greater than 1 yr of age had either unilateral ovariectomy (ULO) or control surgery. Irregular estrous cycles and periods of constant estrus were more frequent during a period of 90 days after ULO than in controls. Follicle-stimulating hormone (FSH) concentration in plasma collected at 0900-1100 h of the metestrus nearest to 20, 50, and 90 days after surgery was increased by ULO; in both treatment groups, FSH increased between 20 and 90 days. Compensation in ovarian weight and number of corpora lutea had occurred by 90 days after ULO. Estradiol, estrone and luteinizing hormone (LH) concentrations did not change with time or treatment. Numbers of small, medium and large antral follicles per ovary at metestrus were increased by ULO, while the number of follicles per rat was decreased. It was concluded that the reduction in ovarian tissue (which decreased the number of growing follicles) resulted in an elevation of basal FSH followed by irregularity in estrous cycles.     

Full-term development of rats from oocytes fertilized in vitro using cryopreserved ejaculated sperm

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen–thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen–thawed ejaculated spermatozoa were similar to those of frozen–thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen–thawed ejaculated sperm was slightly increased at 5 h. When the frozen–thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen–thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen–thawed sperm could be developed to term via in vitro fertilization in rats.     

Follicular recruitment in long-term hemicastrate rats

In the long-term hemicastrate rat, the total number of ova shed during estrus is the same as in the intact rat. To determine if the dynamics of follicular development are the same in the hemicastrate rat as in the intact control rat, the remaining ovary was removed from rats 20 to 30 days after hemiovariectomy. Complete serial sections of each ovary were prepared for histological examination. All follicles greater than or equal to 300 micrometers were counted, measured, and examined for signs of atresia. Long-term hemicastrate rats had a total complement of half as many healthy antral follicles compared to intact rats at estrus. At metestrus, there were half as many small and medium antral follicles in long-term hemicastrates as in controls. However, the total number of large antral follicles was the same in hemicastrate and intact rats. Thus, by metestrus, the appropriate number of follicles for ovulation appears to have been achieved in both animals, with all these large antral follicles located in the one remaining ovary of the hemicastrate rat, while they are distributed between both ovaries of the intact rat. Ovaries of the long-term hemicastrate rats contained far fewer attretic follicles than ovaries of intact rats. These findings suggest that the process of follicular recruitment differs greatly between intact and long-term hemicastrate rats. Atresia of small and medium antral follicles (300-400 micrometers in diameter) is apparently a necessary step in achieving the correct number of ovulatory follicles in the intact rat, yet the hemicastrate rat arrives at the correct number of ovulatory follicles without atresia.     

Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5-2.0×1.0-1.2×0.8-1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips.     

Pulpal regeneration and root development after subcutaneous transplantation of cryopreserved immature teeth in rats

The purpose of this in vivo study was to investigate revascularization and root growth after autotransplantation of cryopreserved immature teeth. Immature molar teeth were extracted in 4-week-old Wistar rats. In the test group, teeth were cryopreserved for 1 week and transplanted subcutaneously to the abdomen. In the control group, teeth were transplanted subcutaneously immediately after extraction. Material was collected in test and control animals at intervals of 1, 2, 4 and 10 weeks post-transplantation and histological and microradiographical examination was performed. Results showed that during the first weeks after transplantation, pulpal repair was similar in both groups although degenerated pulpal tissue was replaced slower in cryopreserved teeth and some differences in types of hard tissue formation were found between test and control teeth. After 10 weeks, the differences in the regenerated pulpal tissue between cryopreserved and control teeth observed during the first weeks were no longer detectable. No root growth was detected microradiographically 10 weeks after transplantation in any of the transplanted teeth. The presence of dentin-like tissue in the pulp cavity of some autotransplanted cryopreserved teeth, suggests survival of pulpal tissue after cryopreservation.     

Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.     

Alopecia attributed to neoplastic ovarian tissue in two ferrets

Ferrets with adrenal gland dysfunction have alopecia as their most common clinical sign of disease. Two cases of alopecia in neutered female ferrets are reported that were associated instead with neoplastic tissue found at the site of an ovarian pedicle. Androstenedione and 17-hydroxyprogesterone, but not estradiol, concentrations were high in both ferrets. Following surgical resection of the abnormal tissue in one ferret, the high hormone values decreased quickly and hair regrowth ensued. In both cases, histologic examination revealed features consistent with classical sex cord-stromal (gonadostromal) tumors: prominent spindle cells, along with polyhedral epithelial cells and cells with vacuolated cytoplasm. Although similiar cell types have been described in the adrenal glands of ferrets with adrenal-associated endocrinopathy, an ovarian origin for the current neoplasms is considered likely on the basis of their anatomic location; accessory adrenal tissue has only been described close to an adrenal gland or in the cranial perirenal fat of ferrets. Immunohistochemical analysis, using an antibody against Mullerian-inhibiting substance, failed to prove definitively the source of the steroidogenic cells.     

Damage to ovarian development and function

Ovarian function in women can be compromised by exposure to toxic environmental factors. Chemicals that affect ovarian function can act through direct effects on hormone action (ovary) or by interference with steroid hormone action (hypothalamus and/or pituitary). These effects can cause problems in the form of infertility. Alternatively, ovarian toxicants can directly cause ovarian failure by extensive follicular destruction. This targeting can result in loss of ovarian steroid hormones, eventual ovarian failure (menopause), and ultimate disruption of neuroendocrine feedback causing increased levels of FSH and LH. This article provides an overview of chemicals that in animal studies have been identified to cause disrupted ovarian function with a focus on the sites of targeting by which these disruptions occur. In predicting the impact of environmental factors on reproductive function in women, it is critical to gain a better appreciation of the physiological consequences resulting from the potential variety of mechanisms by which toxicants can disrupt ovarian function. This article attempts to provide such a perspective within the context of specific chemicals for which ovarian sites of toxicity have been identified.This work was supported by ES08979, ES09246, AG021948, Center Grant ES06694.