Canine Ovarian Tissues Research Articles

Cryopreservation of canine ovarian tissue by slow freezing and vitrification: Evaluation of follicular morphology and apoptosis rate

In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.

The spatio-temporal distribution of aromatase cytochrome in ovary throughout the canine oestrous cycle.

New animal welfare legislation and ethical guidelines encourage alternative approaches for canine contraception, instead of surgical gonadectomy which is considered invasive and unjustified in healthy dogs. Reversible contraception might be achieved by inhibition of aromatase (CYP19), an enzyme catalysing the conversion of androgens to oestrogens. This study provides insights into the spatio-temporal expression and distribution of aromatase in canine ovarian tissue. Ovarian tissue was collected from 39 healthy and sexually mature bitches during different stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10). Localisation of cytochrome P450 aromatase was determined by immunohistochemistry. Aromatase activity in the dog is high during pro-oestrus, ovulation and early dioestrus. Comparing types of follicles and corpora lutea, the highest aromatase abundance was found in antral follicles and luteinising follicles, whereas corpora lutea and early antral follicles showed an intermediate presence of the enzyme. Interesting was the high abundance of aromatase in luteinising theca interna cells, prevailing over granulosa cells. Understanding of cells involved in oestradiol production is important for targeted inhibition of oestradiol synthesis, possibly offering an approach for contraception and suppression of oestrus.

Effects of different subcutaneous sites on heterotopic autotransplantation of canine ovarian tissue.

Ovarian tissue transplantation makes it possible to restore fertility; however, the success of this technique depends on the transplant region used. Therefore, this study aimed to evaluate the effect of two subcutaneous regions on canine ovarian transplantation, pinna (Pi) and neck (Ne), for 7 and 15 days. Ovaries collected by ovariosalpingohysterectomy were fragmented using a punch device. Fresh fragments were fixed, and the others were immediately grafted onto the animal itself in the Pi and Ne regions for 7 and 15 days. Recovered fragments were evaluated for histology (morphology, development and stromal density), picrosirius (collagen fibers), and immunohistochemistry (fibrosis and cell proliferation). The results showed that follicular normality rates were lower in Pi-7 (78%) vs. control (90%) and Pi-15 (86%), similar in Ne-7 (92%) and superior in Ne-15 (97%) compared to the control, with the effect of the region Ne (94%) superior (P < 0.05) to Pi (82%). Stromal density reduced in both regions vs. control but was similar within 15 days. Fragments from both regions showed higher fibronectin labeling and deposition of type I and lower type III collagen fibers (P < 0.05) vs. control. Proliferation rates in Ne-7 were higher (P < 0.05) than in control, and Pi-15 was higher (P < 0.05) than Ne-15. In conclusion, the pinna may be a region with greater potential than the neck after a 15-day autotransplantation of canine ovarian tissue.

Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device.

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5%±1.6% vs. VIT: 52.05%±1.5%). Although the rates of normal primordial (71.1%±1.8%) and secondary (0.7%±0.4%) follicles vitrified showed a reduction (p<.05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1%±1.5% and 2.3%±0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65±12.8 vs. VIT: 199.68±10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.

Vitrification of canine ovarian tissues with polyvinylpyrrolidone preserves the survival and developmental capacity of primordial follicles

Ovarian tissue cryopreservation combined with immature follicle development can preserve female fertility in wildlife, regardless of age or reproductive timing. To investigate the effects of different cryopreservation methods and cryoprotectants on follicular survival and developmental capacity, ovarian cortical pieces from 15 dogs were cryopreserved by slow freezing or vitrification with different additional cryoprotectants as follows: dimethyl sulfoxide (DMSO), polyvinylpyrrolidone (PVP), combined DMSO and PVP (each at half the concentration of when used independently), or none (control). Cryopreserved ovarian tissues were evaluated by neutral red staining, histology, and xenotransplantation assays. Among cryopreservation conditions tested, vitrification with combined DMSO and PVP significantly improved the maintenance of follicular morphology compared to that in control. Furthermore, ovarian tissues vitrified using this condition maintained follicle morphology and developmental capacity 9 weeks after grafting, as shown by an increased percentage of primary and secondary follicles and a significant decrease in the transition stage from primordial to primary stage follicles 5 and 9 weeks after grafting. In contrast, slow freezing and control groups lost intact follicles by 5 weeks after grafting. The described cryopreservation techniques, which preserve canine follicle development, will build the foundation of ovarian tissue cryopreservation to preserve female fertility in wild canids.

Mesenchymal-like stem cells in canine ovary show high differentiation potential.

Recent studies have reported the existence of stem cells in ovarian tissue that show enhanced proliferative and differentiation potential compared to other adult tissues. Based on this evidence, we hypothesized that ovarian tissue contained mesenchymal-like stem cells (MSC) that could be isolated using a novel rapid plastic adhesion technique. We established MSC lines derived from ovarian and adipose tissue based on their ability to rapidly adhere to plastic culture dishes in the first 3hours after plating and studied their potentiality in terms of molecular markers and differentiation capacity. Morphological and kinetic properties of in vitro cultured ovarian MSC were similar to adipose-derived MSC, and both reached senescence after similar passage numbers. Ovarian-derived MSC expressed mesenchymal (CD90 and CD44) but not haematopoietic markers (CD34 and CD45), indicating similarity to adipose-derived MSC. Moreover, ovarian-derived MSC expressed NANOG, TERT, SOX2, OCT4 and showed extensive capacity to differentiate not only into adipogenic, osteogenic and chondrogenic tissue but also towards neurogenic and endodermal lineages and even precursors of primordial germ cells. These results show for the first time the derivation of ovarian cells with the molecular properties of MSC as well as wide differentiation potential. Canine ovarian tissue is accessible, expandable, multipotent and has high plasticity, holding promise for applications in regenerative medicine.

Distribution of follicles in canine ovarian tissues and xenotransplantation of cryopreserved ovarian tissues with even distribution of follicles.

Ovarian follicles are not homogeneously distributed within the ovarian cortex in several species of mammals. Yet to maximize the reproducibility of experimental results of ovarian transplantation, it is essential to assess the degree of density and distribution of follicles in ovarian tissues before their transplantation. In this study, the ovarian cortex from 13 immature bitches (ten purebred and three mongrels) was sectioned into 1.0- to 1.5-mm3 cubes, those were fixed, sectioned and stained with haematoxylin and eosin. To evaluate the density and distribution of follicles, the mean number of all stages of follicles per square millimetre was calculated after observation under a microscope. The distribution of follicles was considered even when the variance value was lower than 10 or between 10 and 16, with an absolute value of distortion inferior to 1. The mean number of follicles ranged from 3.24 to 28.34/mm2 in 25 ovaries from 13 bitches examined. The variance and distortion ranged from 0.35 to 119.64 and -1.87 to 4.40, respectively. The distribution of follicles within the ovarian cortex was judged uneven in 12 of 25 ovaries. These results indicated that follicles were not homogeneously distributed within the ovarian cortex in a large proportion of ovaries. In addition, cryopreserved ovarian fragments with even distribution of follicles were transplanted to NSG mice with or without 400 U/kg of disialylated erythropoietin (asialo EPO). After removing both sides of ovary, a piece of ovarian fragment was placed under the kidney capsule in both sides of kidney. At 4weeks after transplantation, the fragments were recovered from the mice and the number of primordial, primary, secondary and antral follicles was counted. Total number of follicles and survival rates of follicles in transplanted fragments with asialo EPO were higher than without asialo EPO in four bitches examined. These findings suggest that asialo EPO might be effective on the follicular survival of canine ovarian tissues after xenotransplantation. Knowing the degree of density and distribution of follicles in ovarian tissues before transplantation is expected to contribute to the precise interpretation of results after transplantation of the ovarian tissues.

Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Abstract: Although vitrification and follicular survival subsequent to xenotransplantation of canine ovarian tissues have been reported, the degree of cryoinjury after the vitrification has not yet been fully examined. Since cryoinjury after vitrification of ovaries has been reported in bovine and humans, this study evaluated canine ovarian tissues after vitrification by immunohistochemistry and TUNEL assay. Ovaries were collected from immature bitches. The ovarian tissue cubes were pretreated with 1 M DMSO. Subsequently, DAP 213 solution (2 M DMSO, 1 M acetamide, 3 M propylene glycol) was added. The cryotubes containing the ovarian tissues were placed on ice for 5 min, then plunged directly into liquid nitrogen. After warming with 0.25 M sucrose, cryopreserved ovarian tissues were fixed and subsequently stained with hematoxylin-eosin, proliferation cell nuclear antigen (PCNA) antibody, or active caspase-3 (AC-3) antibody, and evaluated by the TUNEL assay. There were no differences in the numbers of primo...

Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Cryopreservation of canine ovarian cortex using DMSO or 1,3-propanediol

Cryopreservation of ovarian cortex is potentially an important tool for the conservation of endangered species. It will allow preserving the large pool of primordial and primary follicles to retrieve fertilizable oocytes in the future. The aim of this study was to evaluate the effects of slow freezing on the morphology and viability of canine follicles after thawing using DMSO or 1,3-propanediol (PROH) as cryoprotectants. Slices of canine ovarian tissue were equilibrated for 20 minutes at 20 °C in minimum essential medium containing either cryoprotectants at 1.5 M, and then frozen by a standardized protocol. Morphology of follicles after thawing was analyzed by means of histology and transmission electron microscopy, and viability was assessed using Trypan blue and fluorescent probes. The exposure of dog ovarian tissue to both cryoprotectants before freezing had no effect on follicular morphology and viability. Also after freezing, follicles remained histologically normal, but transmission electron microscopy revealed damage of ultrastructure in follicles, which were exposed to PROH. Postthaw viability was significantly reduced with 65.7% of the follicles remaining alive in DMSO and 48.7% in PROH. In conclusion, this study demonstrated the survival of canine oocytes within ovarian cortex cryopreserved by slow freezing using 1.5-M DMSO.

Immunolocalization of Progesterone Receptors in the Canine Oviduct around Ovulation

In the bitch, oocyte maturation, sperm storage, fertilization and early embryo development take place within the oviducts under high and increasing circulating progesterone concentrations. To investigate the potential effects of progesterone on the canine oviduct, nuclear progesterone receptors (PR) were localized. Oviducts were collected by ovariectomy from adult Beagle bitches during anestrus, after the onset of proestrus but prior to the Luteinizing Hormone (LH) peak (Pre-LH), after the LH peak but before ovulation (Pre-ov) and on Days 1, 4 and 7 after ovulation (n = 3 bitches per stage). The cellular distribution of PR was studied by immunohistochemistry (IHC) in the ampulla, isthmus and tubal part of the utero-tubal junction (UTJ). Plasma progesterone and 17β-oestradiol were assayed on the day of surgery. PR were specifically expressed in the nuclei of epithelial, stromal and muscular cells in the ampulla, isthmus and UTJ. The IHC scores did not vary from one oviductal region to another. However, the epithelium displayed higher scores than the stroma at anestrus, Pre-ov, Days 4 and 7, and also higher scores than muscle at Days 4 and 7 (p < 0.05). Immunohistochemistry scores in the stroma and muscle decreased at Days 4 and 7 compared with previous stages (p < 0.05). Furthermore, muscular IHC scores were positively correlated with circulating 17β-oestradiol concentrations and negatively correlated with circulating progesterone concentrations (p < 0.05). In conclusion, PR were identified in the canine oviduct, with differences in expression between tissues and times around ovulation, suggesting that progesterone may regulate tubal functions and reproductive events in this species.

Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse

The present study evaluated: (1) in vivo follicular development in canine ovarian tissue after slow freezing and xenotransplantation; and (2) the use of erythropoietin (EPO) as an angiogenic factor to optimise the transplantation procedure. Frozen-thawed ovarian tissue from five bitches was grafted into severe combined immunodeficient (SCID) mice (n=47) treated with or without EPO (500 IU kg(-1), once daily for 3 days) (Groups A and B, respectively) and analysed after 0, 1, 8 or 16 weeks. Follicle grade, follicle density, follicle morphology and stromal cells density were assessed by histological analysis, whereas vascularisation of the graft was quantified by immunohistochemistry with anti-α-smooth muscle actin antibody. Despite a massive loss of follicles after grafting, secondary follicle density was higher at 8 and 16 weeks than at 1 week regardless of EPO treatment. EPO significantly improved early follicle morphology and stromal cell density after 8 weeks and blood vessel density at 16 weeks after transplantation (P<0.05). Intact secondary follicles with more than three granulosa cells layers were observed 16 weeks after transplantation. The results suggest that canine ovarian tissue can be successfully preserved by our slow-freezing protocol because the tissue showed follicular growth after xenotransplantation. EPO treatment did not lessen the massive loss of follicles after transplantation.

Effect of Intraperitoneal Administration of Desialylated Erythropoietin on the Follicular Survival in Cryopreserved Canine Ovaries after Xenotransplantation

The effect of intraperitoneal administration of desialylated erythropoietin (asialo EPO) on the follicular reserve of cryopreserved canine ovarian tissues was examined using non obese diabetic-severe combined immunodeficient (NOD-SCID) mice was examined. Vitrified-warmed canine ovarian tissues were placed under the dorsal skin of mice, and were then treated with saline or 400 or 4,000 U/kg/day of asialo EPO by intraperitoneal injection from one day before transplantation for 5 days. Transplanted tissues were removed and subjected to a histological examination 4 weeks after the transplantation. Without asialo EPO treatment, the mean number of primordial follicles in the cryopreserved ovarian tissues had decreased from 4.3/mm2 to 0.2/mm2 at 4 weeks after xenotransplantation. However, higher numbers of follicles were observed when recipient mice were treated with asialo EPO. The average number of primordial follicles per square millimetre of ovarian section was 0.2 in the saline group and 1.1 in the 400 U/kg/day of asialo EPO at 4 weeks after transplantation. These results indicate that the systemic administration of asialo EPO could be effectively used to enhance the survival of the follicles of transplanted cryopreserved canine ovaries.

Follicle Formation in the Canine Ovary After Autografting to a Peripheral Site

This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.

Follicular Loss of the Cryopreserved Canine Ovary after Xenotransplantation

Abstract: The effect of cryopreservation and subsequent xenotransplantation on the follicular reserve of the canine ovary by using non obese diabetic-severe combined immunodeficient (NOD-SCID) mice was examined. Vitrified-warmed canine ovarian tissues were placed into the ovarian bursa of mice, and then were removed and subjected to histological examination at 4 weeks after the transplantation. Over 30% of primordial follicles and 65% of early primary follicles survived after cryopreservation. However, regardless of breed or age, percentages of survived primordial follicles and early primary follicles after the transplantation ranged from 0–7% and from 0–15%, respectively. These results indicate that the majority of primordial follicles and early primary follicles in vitrified-warmed canine ovarian tissues disappear after xenotransplantation. Further studies will be required to be able to enhance the survival of transplanted cryopreserved ovarian follicles in canine.

Vitrification of Canine Embryos at Various Developmental Stages.

The cryopreservation of the genetic resource materials such as oocytes and embryos can be useful for improved breeding of companion and working dog, including guide dog for the blind. Vitrification has been widely developed to apply to cryopreservation of mammalian embryos. In the mouse, vitrification of the embryos was succeeded by DAP213 method, moreover, it was available to vitrify the canine ovarian tissues. While, porcine oocytes were vitrified successfully using the E30S method. However, the suitability of both vitrification methods for canine embryos has not been investigated. To improve the breeding management programs in guide dogs for the blind, the objective of the present study was to compare DAP213 with E30S method for vitlification of the embryos at 1-cell to blastocyst stages. Female Labrador Retrievers were inseminated on day 4-6 after LH surge. On day 10-15 after LH surge (day 4-10 after insemination), embryos were collected from excised oviducts and uteri of bitches by flushing, and were vitrified DAP213 or E30S method. In the DAP213 method, embryos were transferred into a cryotube containing 1 M DMSO. Subsequently, DAP213 solution, composed of 2 M DMSO, 1 M acetamide and 3 M propylene glycol, was added to each cryotube. After placing the cryotube in ice water for 5min, they were plunged directly into liquid nitrogen. In the E30S method, the embryos were exposed to PB1 containing 5, 10 and 20% ethylene glycol (EG), and 30% EG supplemented with 0.5 M sucrose for 5, 2, 2 and 1 min, respectively, at room temperature (23 ± 2 °C). They were then placed on a cryotop sheet (Kitazato Supplies, Japan), and the cryotop was immediately plunged into liquid nitrogen. After thawing, embryos were stained with propidium iodide for assessment of plasma membrane integrity of blastomeres. The blastomeres with disrupted plasma membrane were dyed red with PI. The E30S method showed fewer morphological abnormalities of vitrified embryos than the DAP213 method, and a few abnormalities of vitrified embryos in which integral blastomeres accounted for more than 75% at 1-cell to morula stages. However, blastomeres were mostly damaged at blastocyst stage embryos in the E30S group. While, almost blastomeres of vitrified embryos in the DAP213 group had disrupted plasma membrane regardless of developmental stage. These results suggest that the E30S method of vitrification is suitable for cryopreservation of canine embryos, and embryos at earlier developmental stage have a high tolerance to cryopreservation comparing with them at blastocyst stage. Although further study seems to be required to develop an optimal cryopreservation method for canine blastocysts and it is necessary to produce the pups derived from vitrified embryos, our findings may contribute to the efficient production of guide dogs for the blind.

Biosynthesis of the canine zona pellucida requires the integrated participation of both oocytes and granulosa cells.

In the dog, attempts to localize the expression of zona pellucida (ZP) proteins during folliculogenesis have failed to demonstrate conclusively whether any or all of the zona proteins are synthesized in the oocyte or the granulosa cells. Probing of paraformaldehyde-fixed prepubertal canine ovarian tissue sections with a panel of fluorescently conjugated lectins localized the expression of glycoproteins during folliculogenesis. We confirm that six lectins (PSA, s-WGA, ECL, GSL-II, LEL, and STL) consistently labeled the ZP and adjacent granulosa cells of the developing follicle and that canine ZP expresses beta-gal(1,4)glcNAc, beta-gal(1,3)galNac, alpha-mannose, and terminal sialic acid residues in a developmentally specific manner. Riboprobes for canine ZPA and ZPC genes were produced and used for in situ hybridization studies of mRNA expression in canine folliculogenesis. In addition, we isolated a partial cDNA transcript from total ovarian RNA for the canine ZPB gene having a high degree of sequence identity with the felid and porcine ZPB homologues. Subsequently, the ZPA gene transcripts were localized to the cytoplasm of oocytes in primordial, primary, and early secondary follicles. We then localized expression of ZPB and ZPC gene transcripts to the granulosa cells of growing follicles, but not in squamous granulosa cells of primordial follicles or oocytes. These observations indicate that in the juvenile canine ovary, the oocyte is responsible for synthesis of the ZPA protein and directing synthesis of the ZPB and ZPC proteins by the granulosa cells and that ZP gene transcription occurs in a sequential manner during folliculogenesis.