Responsiveness of intraovarian dog follicles in vitro to epidermal growth factor and vascular endothelial growth factor depends on ovarian donor age.

We investigated the influence of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) on in vitro follicle development within ovarian cortices recovered from pre-pubertal (≤6 months) versus peri-pubertal dogs (≥10 months). Ovarian cortices were cultured for 3 or 7 days in EGF (0 or 10 ng/ml) and VEGF (0, 0.1 or 1 ng/ml) and subjected to histological and apoptosis analyses. Fresh cortices from the same dogs served as "non-cultured controls" (NCC) and were evaluated similarly. The response of ovarian follicles to growth factors differed between pre-pubertal versus peri-pubertal tissues. For pre-pubertal dogs, percentage of structurally normal follicles in cortices cultured for 3 days in low VEGF (0.1 ng/ml) and EGF alone was comparable to that of the NCC. Follicle density declined in all cultured groups even after 3 days. Primary follicle diameter in all cortices cultured for 7 days, except in low VEGF, was smaller than that of the NCC, and percentage apoptotic follicles sharply increased in all treatment groups compared to the NCC. For peri-pubertal donors, percentages of structurally normal follicles decreased in all culture treatments at 3 and 7 days of incubation compared to the NCC. However, more normal follicles were found in cortices cultured in low VEGF and the two VEGF and EGF combinations than in the absence of growth factors or with EGF alone. Culture reduced the density of developing follicles, but follicle diameter was similar to that of the NCC. TUNEL analysis revealed that high-VEGF (1 ng/ml) treatment protected follicles against apoptosis, with the proportion of apoptotic follicles at Day 7 being comparable to that of the NCC. The findings demonstrate that the response of ovarian cortices to growth factor supplementation varied between pre-pubertal versus peri-pubertal donors.

Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.

Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.

Endocrine and paracrine controls of canine follicular development and function.

Canid reproduction is unique among other mammals in that females experience long and variable periods of ovarian inactivity. While the domestic dog exhibits a non-seasonal, largely sporadic monoestrus occurring once or twice a year, most wild canids, such as the gray wolf (Canis lupus) and red wolf (Canis rufus), are seasonal breeders with onset apparently dependent on species, latitudinal location and/or variety of environment factors. Neuroendocrine controls of ovarian functions have been mostly studied in the dog, but less so in their wild counterparts, due to difficulties in regular blood sampling. Yet, development of non-invasive hormone monitoring has advanced the understanding of reproductive cycle in wild canids. Recent advances in in vitro follicle culture technology also have begun to provide insights into paracrine controls of canid ovarian folliculogenesis. This review highlights current knowledge on canid reproduction with emphasis on endocrine and paracrine controls of follicular development. We also discuss future research priorities, including advancing the understanding of anoestrous termination and role of paracrine factors in canine folliculogenesis.

Dynamic changes in mitochondrial DNA, distribution and activity within cat oocytes during folliculogenesis.

Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial (n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral (n = 44), early antral (n = 66), small antral (n = 59), antral (n = 41) and advanced antral (n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mtDNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.

Birth of kittens after the transfer of frozen-thawed embryos produced by intracytoplasmic sperm injection with spermatozoa collected from cryopreserved testicular tissue.

The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection (ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post-ICSI, presumptive zygotes/cleaved embryos were treated with 10 µm forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/hCG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm.