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PURPOSE: Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. METHODS: Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. RESULTS: No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. CONCLUSIONS: Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

Remerciements : Pôle STAR; Plateforme CIRE, INRA, UMR PRC 0085, Centre Tours Val de Loire; While cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOC) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse-DOC was obtained, regardless of maturation media (complex, semi-defined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. DOC subjected to parthenogenetic activation (PA) had greater (P

The aim of the present study was to establish the presence of BMP type I and II receptor presence in preantral follicles and to investigate the effect of BMP4 supplementation on preantral follicle activation and development during organotypic culture of prepubertal ovine ovarian cortex pieces. Ovine ovarian fragments were cultured with varying concentrations (0, 25, 50 or 100 ng/ml) of BMP4 in the presence or absence of FSH in the culture media to determine the optimal minimum dose for preantral follicle activation and development. Follicular morphometry, immunohistochemistry for BMPR-IA, BMPR-IB, BMPR-II, proliferating cell nuclear antigen and TUNEL progesterone and 17β-oestradiol production were assessed. Follicle and oocyte diameter were positively influenced by the addition of BMP4 to culture. However, there was no effect of BMP4 on primordial follicle activation. Treatment with BMP4 reduced progesterone synthesis but had no effect on oestradiol. The percentage of primordial follicles stained with TUNEL was significantly greater in the culture without BMP4 compared to other culture systems after 9 days of culture. There was an increase in PCNA immunoreactivity in all follicles regardless of treatment. Treatment with BMP4 increases the size of oocytes and follicles but has minimal effects on follicular dynamics during in vitro culture of ovarian cortical tissue of sheep.

The process of folliculogenesis is a complex and dynamic process that is regulated by positive and negative factors. During folliculogenesis in the mammalian ovary, the primordial follicle pool is gradually reduced through successive waves of follicle growth. It is important that this pool is carefully regulated, otherwise premature ovarian failure will occur. This process results in the ovulation of an ovocyte or more commonly, the loss of ovocytes through atresia. The majority of research has concentrated on the positive regulators of follicle growth with very little attention on the negative regulators. Nonetheless, there are several factors that have been observed to inhibit follicle activation and development. In this review, we summarize those here.

Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P

STUDY HYPOTHESIS Is the c-Jun-N-terminal kinase (JNK) pathway implicated in primordial follicle activation? STUDY FINDING Culture of ovine ovarian cortex in the presence of two different c-Jun phosphorylation inhibitors impeded pre-antral follicle activation. WHAT IS KNOWN ALREADY Despite its importance for fertility preservation therapies, the mechanisms of primordial follicle activation are poorly understood. Amongst different signalling pathways potentially involved, the JNK pathway has been previously shown to be essential for cell cycle progression and pre-antral follicle development in mice. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovine ovarian cortex pieces were cultured with varying concentrations of SP600125, JNK inhibitor VIII or anti-Mullerian hormone (AMH) in the presence of FSH for 9 days. Follicular morphometry and immunohistochemistry for proliferating cell nuclear antigen (PCNA), apoptosis and follicle activation (Foxo3a) were assessed. MAIN RESULTS AND THE ROLE OF CHANCE Inhibition of primordial follicle activation occurred in the presence of SP600125, JNK inhibitor VIII and AMH when compared with controls (all P < 0.05) after 2 days of culture. However, only in the highest concentrations used was the inhibition of activation associated with induction of follicular apoptosis (P < 0.05). In growing follicles, PCNA antigen expression was reduced when the JNK inhibitors or AMH were used (P < 0.05 versus control), indicating reduced proliferation of the somatic compartment. LIMITATIONS, REASONS FOR CAUTION Although we evaluated the effects of inhibition of c-Jun phosphorylation on primordial follicle development, we did not determine the cellular targets and mechanism of action of the inhibitors. WIDER IMPLICATIONS OF THE FINDINGS These results are the first to implicate the JNK pathway in primordial follicle activation and could have significant consequences for the successful development of fertility preservation strategies and our understanding of primordial follicle activation. LARGE SCALE DATA n/a. STUDY FUNDING AND COMPETING INTERESTS Dr Michael J. Bertoldo and the laboratories involved in the present study were supported by a grant from 'Region Centre' (CRYOVAIRE, Grant number #320000268). There are no conflicts of interest to declare.

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.

Beyond the potential use of in vitro production of embryos (IVP) in breeding schemes, embryos are also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos, and also benefit assisted procreation in human beings. Efficient in vitro embryo production is currently a major objective for livestock industries, including small ruminants. The heterogeneity of oocytes collected from growing follicles by laparoscopic ovum pick up or in ovaries of slaughtered females, remains an enormous challenge for IVM success, and still limits the rate of embryo development. In addition, the lower quality of the IVP embryos, compared with their in vivo-derived counterparts, translates into poor cryosurvival, which restricts the wider use of this promising technology. Therefore, many studies have been reported in an attempt to determine the most suitable conditions for IVM, IVF, and in vitro development to maximize embryo production rate and quality. This review aims to present the current panorama of IVP production in small ruminants, describing important steps for its success, reporting the recent advances and also the main obstacles identified for its improvement and dissemination.

In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 +/- 1.0 vs. 8.2 +/- 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.

A grade classification (I, II, and III) based on the number of cumulus layers and oocyte morphology is currently used by many laboratories. Oocytes found denuded at collection (grade III) are considered not suitable and routinely discarded. Thus, if a particular strategy could be applied to their use in labour-intensive processes, such as ovum pickup, it would be a benefit. This experiment was performed to examine the effect of IVM medium composition to produce goat embryos in vitro using oocytes already found denuded at collection (DOC). In total, 411 DOC and 141 intact COC (control treatment) obtained by slaughterhouse ovaries were analysed in 4 replicates. The maturation medium consisted in TCM199 supplemented either with (1) 10 ng mL–1 of EGF and 100 µM cysteamine (simplified); (2) 10 ng mL–1 of EGF, 5 IU mL–1 of hCG, 10 IU mL–1 of eCG, 19 ng mL–1 of IGF-1, 2.2 ng mL–1 of FGF, 5 µg mL–1 of ITS, 90 µg mL–1 of l-cystein, 0.1 mM β-mercaptoethanol, 75 µg mL–1 of vitamin C, 720 µg mL–1 of glycine, 0.1 mg mL–1 of glutamine, and 110 µg mL–1 of pyruvate (semidefined); or (3) 10% fetal calf serum (FCS), 100 µM cysteamine, and 50 ng mL–1 of oFSH (complex). Both DOC and COC were subjected to IVM, IVF, and IVD as previously described (Souza-Fabjan et al. 2014, Theriogenology 81, 1021–31). The COC were matured only in simplified medium. On Day 8, all expanded blastocysts were fixed and stained with Hoechst for cell counting. Statistical analysis was performed using all tests with a significant interval of 95%. All variables were compared among treatments using ANOVA and SNK test. The results are described as mean per replicate ± s.e.m. No significant differences were found in simplified, semidefined, or complex medium, respectively, in cleavage rate (52 ± 7.5, 60 ± 9.4, or 51 ± 15.0%), blastocyst from cleaved (36 ± 3.9, 39 ± 9.3, or 41 ± 4.8%), blastocyst from initial DOC (19 ± 5.0, 23 ± 8.1, or 21 ± 3.3%), hatching rate (55 ± 22.9, 55 ± 15.9, or 52 ± 14.8%), or total blastomeres number (184 ± 12.6, 179 ± 12.4, or 190 ± 13.8). The control COC showed no significant differences to any DOC treatment on cleavage (77 ± 3.4%) and blastocyst from cleaved (60 ± 2.2%). However, the blastocyst rate from initial COC was higher (46 ± 0.5%; P < 0.05) than all DOC treatments. Even though the blastocyst yield was lower than COC (~21 v. 46%), it is reasonable to affirm that it is possible to produce embryos from oocytes that would not be utilised, which may represent additional number of embryos. The blastocyst cell numbers in COC (192 ± 13.7) were similar (P > 0.05) to DOC, indicating that the goat embryos produced were of good quality. In conclusion, the inclusion of more complex substances in IVM media did not increase the development rate of DOC and, therefore, more simple IVM media could be used for this purpose. Finally, the goat embryos produced had satisfactorily number of blastomeres, demonstrating that the in vitro development step is able to generate good quality embryos from grade III oocytes. Therefore, some oocytes that in general would be discarded will develop to blastocysts and may represent benefits, especially after ovum pick-up from genetically valuable goats.

Primordial follicles are maintained in a quiescent state until they receive a signal to activate and join the growing pool. It is essential that the rate of follicle activation is well coordinated as it determines the reproductive lifespan of the female. This finely tuned process is under the control of a variety of positive and negative factors, and there is evidence that the JNK pathway is involved. The aim of this study was to assess the effect of a JNK inhibitor (SP600125) on follicle activation in vitro. Ovaries from 6 prepubertal sheep were dissected into 1-mm3 fragments and cultured in the presence or absence of SP600125 (0, 5, or 25 µM; SP0, SP5, and SP25, respectively). After 0, 2, 5, or 9 days, fragments were fixed, sectioned, and analysed by histological morphometry to determine the number and type of follicles in addition to TUNEL analysis for apoptosis. In total, 21 584 follicles were assessed. Follicles were classified as either primordial, intermediate, primary, secondary, or antral. Culture media were also assayed for steroid content. After multinomial regression analysis, there were no differences in the rate of follicle activation between groups on Day 2 of culture. However, after 5 days of culture there were significantly more primordial follicles in SP25 (69 ± 9.15%; P

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.