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Glucocorticoids modulate the feto-maternal interface during the induction of parturition. In the dog, the prepartum rise of cortisol in the maternal circulation appears to be erratic, and information about its contribution to the prepartum luteolytic cascade is scarce. However, the local placental upregulation of glucocorticoid receptor (GR/NR3C1) at term led to the hypothesis that species-specific regulatory mechanisms might apply to the involvement of cortisol in canine parturition. Therefore, here, we assessed the canine uterine/utero-placental spatio-temporal expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1; reduces cortisone to cortisol), and -2 (HSD11B2; oxidizes cortisol to the inactive cortisone). Both enzymes were detectable throughout pregnancy. Their transcriptional levels were elevated following implantation, with a strong increase in HSD11B2 post-implantation (days 18–25 of pregnancy), and in HSD11B1 at mid-gestation (days 35–40) (P

While nonlinear phenomena (NLP) are widely reported in animal vocalizations, often causing perceptual harshness and roughness, their communicative function remains debated. Several hypotheses have been put forward: attention-grabbing, communication of distress, exaggeration of body size and dominance. Here, we use state-of-the-art sound synthesis to investigate how NLP affect the perception of puppy whines by human listeners. Listeners assessed the distress, size or dominance conveyed by synthetic puppy whines with manipulated NLP, including frequency jumps and varying proportions of subharmonics, sidebands and deterministic chaos. We found that the presence of chaos increased the puppy's perceived level of distress and that this effect held across a range of representative fundamental frequency ( f o ) levels. Adding sidebands and subharmonics also increased perceived distress among listeners who have extensive caregiving experience with pre-weaned puppies (e.g. breeders, veterinarians). Finally, we found that whines with added chaos, subharmonics or sidebands were associated with larger and more dominant puppies, although these biases were attenuated in experienced caregivers. Together, our results show that nonlinear phenomena in puppy whines can convey rich information to human listeners and therefore may be crucial for offspring survival during breeding of a domesticated species.

In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered under six aspects: (1) the utero-tubal junction that selects a sperm sub-population entering the oviduct; (2) the presence of sperm binding sites on luminal epithelial cells in the oviduct, which prolong sperm viability and plays a role in limiting polyspermic fertilization; (3) the contractions of the oviduct, which promote sperm migration toward the site of fertilization in the ampulla; (4) the regions of the oviduct, which play different roles in regulating sperm physiology and interactions with oviduct epithelial cells; (5) the time of ovulation, and (6) the steroid hormonal environment which regulates sperm release from the luminal epithelial cells and facilitates capacitation in a finely orchestrated manner., After mating or insemination, the region-specific and hormonally regulated morphology and secretions of the utero-tubal junction and oviduct lead to the selection of a limited sub-population of top quality spermatozoa at the site of fertilization.

Ankyrin proteins (ANKRD) are key mediators linking membrane and sub-membranous cytoskeletal proteins. Recent findings have highlighted a new role of ANKRD31 during spermatogenesis, elucidating its involvement in meiotic recombination and male germ cell progression. Following testicular differentiation, spermatozoa (SPZ) enter into the epididymis, where they undergo several biochemical and enzymatic changes. The epididymal epithelium is characterized by cell-to-cell junctions that are able to form the blood-epididymal barrier (BEB). This intricate epithelial structure provides the optimal microenvironment needed for epididymal sperm maturation. To date, no notions have been reported regarding a putative role of ANKRD31 in correct BEB formation. In our work, we generated an Ankrd31 knockout male mouse model (Ankrd31–/–) and characterized its reproductive phenotype. Ankrd31–/– mice were infertile and exhibited oligo-astheno-teratozoospermia (a low number of immotile SPZ with abnormal morphological features). In addition, a complete deregulation of BEB was found in Ankrd31–/–, due to cell-to-cell junction anomalies. In order to suggest that BEB deregulation may depend on Ankrd31 gene deletion, we showed the physical interaction among ANKRD31 and some epithelial junction proteins in wild-type (WT) epididymides. In conclusion, the current work shows a key role of ANKRD31 in the control of germ cell progression as well as sperm and epididymal integrity.

International audience; Sperm migration through the female genital tract is not a quiet journey. Uterine contractions quickly operate a drastic selection, leading to a very restrictive number of sperm reaching the top of uterine horns and finally, provided the presence of key molecules on sperm, the oviduct, where fertilization takes place. During hours and sometimes days before fertilization, subpopulations of spermatozoa interact with dynamic and region-specific maternal components, including soluble proteins, extracellular vesicles and epithelial cells lining the lumen of the female tract. Interactions with uterine and oviductal cells play important roles for sperm survival as they modulate the maternal immune response and allow a transient storage before ovulation. The body of work reported here highlights the importance of sperm interactions with proteins originated from both the uterine and oviductal fluids, as well as hormonal signals around the time of ovulation for sperm acquisition of fertilizing competence.

International audience; Since the 1970s, rodent and human insulin-secreting pancreatic beta-cell lines have been developed and found useful for studying beta-cell biology. Surprisingly, although the dog has been widely used as a translational model for diabetes, no canine insulin-secreting beta cells have ever been produced. Here, a targeted oncogenesis protocol previously described by some of us for generating human beta cells was adapted to produce canine beta cells. Canine fetal pancreata were obtained by cesarean section between 42 and 55 days of gestation, and fragments of fetal glands were transduced with a lentiviral vector expressing SV40LT under the control of the insulin promoter. Two Lox P sites flanking the sequence allowed subsequent transgene excision by Cre recombinase expression. When grafted into SCID mice, these transduced pancreata formed insulinomas. ACT-164 is the cell line described in this report. Insulin mRNA expression and protein content were lower than reported with adult cells, but the ACT-164 cells were functional, and their insulin production in vitro increased under glucose stimulation. Transgene excision upon Cre expression arrested proliferation and enhanced insulin expression and production. When grafted in SCID mice, intact and excised cells reversed chemically induced diabetes. We have thus produced an excisable canine beta-cell line. These cells may play an important role in the study of several aspects of the cell transplantation procedure including the encapsulation process, which is difficult to investigate in rodents. Although much more work is needed to improve the excision procedure and achieve 100% removal of large T antigen expression, we have shown that functional cells can be obtained and might in the future be used for replacement therapy in diabetic dogs.

Our objectives were to describe, in Beagle dogs, the ontogenesis of beta (insulin-producing) and alpha (glucagon-producing) cells from fetal to early postnatal life and adulthood. In addition, to have some insight into interspecies comparison, Beagle dog pancreases were compared to pancreases from a Labrador and Chow Chow. At midgestation, the epithelium was dense, beta cells scarce, and alpha cells numerous and concentrated in the center of the pancreatic bud. From 36 to 45 days post conception (pc), beta cell numbers increased and the epithelium expanded and branched out. At 55 days pc, large beta cell aggregates were seen. At weaning, the islets were similar to those in adults, with limited alpha cells intermingled with numerous beta cells. Quantification of the Alpha to Beta cells ratio has shown a gradual increase of beta cells proportion throughout development. Similar findings were obtained in the two other breeds. In conclusion, in the fetal Beagle dog beta cells emerge from the pancreatic bud at midgestation, but the endocrine structure is mature only in early postnatal life. The ontogenesis of the endocrine pancreas demonstrated in dogs resembles that reported in rats and mice. In contrast, human beta cells appear earlier, at the beginning of the second trimester of gestation. Our study provides a detailed morphological description of pancreatic development in dogs but supplies no information on alpha- or beta-cell function during fetal life. The morphological data reported here provide a foundation for building physiological studies.

International audience; In species where semen is deposited in the vagina, the cervix has the unique function of facilitating progress of spermatozoa towards the site of fertilisation while also preventing the ascending influx of pathogens from the vagina. For the majority of species, advances in assisted reproduction techniques facilitate the bypassing of the cervix and therefore its effect on the transit of processed spermatozoa has been largely overlooked. The exception is in sheep, as it is currently not possible to traverse the ovine cervix with an inseminating catheter due to its complex anatomy, and semen must be deposited at the external cervical os. This results in unacceptably low pregnancy rates when frozen-thawed or liquid stored (> 24 h) semen is inseminated. The objective of this review is to discuss the biological mechanisms which regulate cervical sperm selection. We assess the effects of endogenous and exogenous hormones on cervical mucus composition and discuss how increased mucus production and flow during oestrus stimulates sperm rheotaxis along the crypts and folds of the cervix. Emerging results shedding light on the sperm-cervical mucus interaction as well as the dialogue between spermatozoa and the innate immune system are outlined. Finally, ewe breed differences in cervical function and the impact of semen processing on the success of fertilisation, as well as the most fruitful avenues of further investigation in this area are proposed.

International audience; During ejaculation and the deposition in the female genital tract, spermatozoa undergo hypo-osmotic stress and need to withstand it for optimal fertility. Resistance to hypo-osmotic stress may be affected by the interaction of the spermatozoa with seminal fluid components. The hypo-osmotic resistance of epididymal and ejaculated spermatozoa from dogs, rams and boars was assessed by flow cytometric measurement of sperm viability after incubation in NaCl solutions with osmolalities ranging from 0 to 300 mmol/kg. The hypotonic resistance of epididymal spermatozoa was greater than those of ejaculated spermatozoa in all three species. Among species comparison revealed that ejaculated spermatozoa from dogs were much more resistant than those from rams and boars as 80.4 ± 5.3%, 56.7 ± 4.7 and 9.6 ± 3.6% of live spermatozoa were observed following exposure to an osmolality of 90 mmol/kg in dogs, rams and boars respectively. This can be explained by the fact that dog, ram and boar differ markedly in composition of the seminal plasma owing to the presence (ram, boar) or absence (dog) of seminal vesicles. Hypotonic resistance of epididymal and ejaculated dog spermatozoa was similar whereas ram and boar spermatozoa showed a marked drop in resistance after ejaculation. The in vitro incubation of boar epididymal spermatozoa with raw seminal plasma or the seminal plasma protein fraction induced a similar loss of resistance, suggesting that seminal proteins are involved in the lack of resistance to hypotonic stress of boar ejaculated spermatozoa.

From many endangered or threatened species which are expected to profit from assisted reproduction techniques, mainly epididymal sperm of dead or freshly castrated males are available. These sperm had contact to epididymal secretion products but not to seminal fluid components. Notably, products of accessory sex glands have been shown in domestic animals to condition sperm for fertilization, in particular by mediating sperm-oviduct interaction. We report for the first time that motile epididymal sperm from domestic cats are able to bind to fresh oviduct epithelial cell explants from preovulatory females (median [min, max] of 10 [8, 16] and 10 [8, 17] sperm per 0.01 mm(2) explant surface from both isthmic and ampullar regions, respectively). More sperm attach to the explants when epididymal sperm were preincubated for 30 minutes with seminal fluid separated from electroejaculates of mature tomcats (median [min, max] of 17 [13, 25] and 16 [12, 21] sperm per 0.01 mm(2) explant surface from isthmus and ampulla, respectively). The proportion of bound sperm increased from a median of 54% to 62% by seminal fluid treatment. Sperm-oviduct binding could be facilitated by the decelerated sperm motion which was observed in seminal fluid-treated samples or supported by seminal fluid proteins newly attached to the sperm surface. Seminal fluid had no effect on the proportion of sperm with active mitochondria. Extent and pattern of sperm interaction in vitro were independent of explant origin from isthmus or ampulla. Sperm were attached to both cilia and microvilli of the main epithelial cell types present in all explants. In contrast to published sperm-binding studies with porcine and bovine oviduct explants where predominantly the anterior head region of sperm was attached to ciliated cells, the tails of some cat sperm were firmly stuck to the oviduct cell surfaces, whereas the heads were wobbling. Whether this response is a preliminary step toward phagocytosis or a precondition to capacitation and fertilization remains to be determined. In conclusion, treatment of epididymal sperm with seminal fluid or particular protein components should be considered in future investigations for its potential to improve the outcome of artificial insemination in felids.

SUMMARY In the dog, oocyte maturation, fertilization, and early embryo development take place within the oviduct in the presence of increasing circulating progesterone (P4) levels. Expression of the oviduct-specific glycoprotein 1 (OVGP1), known in other species to be estrogen-dependent, was explored by real-time quantitative reverse-transcriptase PCR, Western blotting, and immunohistochemistry in oviducts from adult Beagle bitches during anestrus and at five specific time periods around ovulation: during pro-estrus before the luteinizing hormone (LH) peak (Pre-LH); after the LH peak and before ovulation (Pre-ov); and at Days 1, 4, and 7 after ovulation (n = 6 bitches per stage). Plasma estradiol-17β (E2) and P4 were assayed at all stages. The expression of canine OVGP1 (cOVGP1) was undetectable during anestrus, increased significantly from Pre-LH to Day 1 in parallel with a decrease in plasma E2-to-P4 levels, remained high at Day 4, then decreased at Day 7 in parallel with an increase in plasma P4 levels. In contrast to other mammals, the expression of cOVGP1 was higher in the isthmus than in the ampulla at all stages. In order to explore the potential regulation of cOVGP1 expression by steroids, the 5′-flanking region of the corresponding gene was analyzed for the presence of estrogen- (ERE) and P4-response-element (PRE). An imperfect ERE and three half-ERE were found in this region, but no PREs. In conclusion, cOVGP1 is highly expressed at the time and site of oocyte maturation and fertilization, and is probably under E2 regulation. Further studies are needed to identify the potential roles of cOVGP1 in each process. Mol. Reprod. Dev. 81: 972–982, 2014. © 2014 Wiley Periodicals, Inc.

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.

Canine oocyte maturation and fertilization take place within the oviducts under increasing plasma levels of progesterone (P4). In order to investigate the role of P4 in these processes, 51 beagle bitches were treated with the P4 receptor antagonist aglepristone at the end of proestrus and 32 females were kept untreated. Fifteen treated and 13 control bitches were inseminated at Days +1 and +2 after ovulation (Day 0). Stages of oocyte maturation and embryo development were determined after ovariectomy at different time points after ovulation. Aglepristone did not prevent ovulation but delayed the resumption of oocyte meiosis and inhibited its progression: first metaphase I (MI) stage was observed at 173 h postovulation and 39% of oocytes reached MII as late as 335 h postovulation in treated females whereas first MI occurred at 76 h and 100% of oocytes were in MII at 109 h postovulation in controls. Aglepristone extended the stay of morphologically normal oocytes within the oviducts: first signs of oocyte degeneration were observed at 335 h in treated versus 100- to 110-h postovulation in control bitches. In inseminated females, aglepristone prevented sperm progression toward the oviducts and fertilization, although motile spermatozoa were observed in the uterine tip flush and within the cranial uterine glands. A proteomic analysis of the tubal fluid from treated and control noninseminated bitches at Day +4 found evidence of 79 differential proteins potentially involved in the oocyte phenotype. In conclusion, P4 plays key roles in postovulatory canine oocyte maturation, aging, and in fertilization.

International audience; Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.

International audience; At the gastrula phase of development, just after the onset of implantation, the embryo proper is characterized by extremely rapid cell proliferation. The importance of DNA repair is illustrated by embryonic lethality at this stage after ablation of the genes involved. Insight into mutation induction is called for by the fact that women often do not realize they are pregnant, shortly after implantation, a circumstance which may have important consequences when women are subjected to medical imaging using ionizing radiation. We screened gastrula embryos for DNA synthesis, nuclear morphology, growth, and chromosome aberrations (CA) shortly after irradiation with doses up to 2.5 Gy. In order to obtain an insight into the importance of DNA repair for CA induction, we included mutants for the non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, as well as Parp1-/- and p53+/− embryos. With the pUR288 shuttle vector assay, we determined the radiation sensitivity for point mutations and small deletions detected in young adults. We found increased numbers of abnormal nuclei 5 h after irradiation; an indication of disturbed development was also observed around this time. Chromosome aberrations 7 h after irradiation arose in all genotypes and were mainly of the chromatid type, in agreement with a cell cycle dominated by S-phase. Increased frequencies of CA were found for NHEJ and HR mutants. Gastrula embryos are unusual in that they are low in exchange induction, even after compromised HR. Gastrula embryos were radiation sensitive in the pUR288 shuttle vector assay, giving the highest mutation induction ever reported for this genetic toxicology model. On theoretical grounds, a delayed radiation response must be involved. The compromised developmental profile after doses up to 2.5 Gy likely is caused by both apoptosis and later cell death due to large deletions. Our data indicate a distinct radiation-sensitive profile of gastrula embryos, including some stage-specific aspects that are not as yet understood.

Compared to other mammals, the canine oocyte offers a very unusual model of meiosis. Its maturation in vitro, studied only over the past ten years, is still poorly controlled : low rate of metaphase II (10 to 20% vs. over 90% in cattle), and high rate of degeneration in cultures (20 to 60%) despite attempts to improve culture media. However, in dogs as well as in canidae threatened by extinction, in vitro maturation is a key step for reproductive biotechnologies, such as in vitro fertilization and embryo production. It is therefore urgent to improve our understanding of the canine oocyte to improve maturation rates. We initiated studies on oocyte maturation in bitches. We examined the role of cAMP in the resumption of meiosis in vitro in bitches, using substances which reduce (Rp-cAMP) or increase (dbcAMP and forskolin) its level inside the oocyte. We also used denuded oocytes to prevent any cAMP supply from the granulosa cells. With this model, we showed that cAMP might play a role in maintaining meiosis, and that the resumption of meiosis may also be controlled by another pathway, possibly involving calcium. Another of our research projects explores changes in the oocyte ultrastructure during in vivo and in vitro maturation. Transmission electron microscopy may provide precise information on possible cytoplasmic anomalies induced by maturation. This fundamental work will eventually help us improve in vitro maturation of canine oocytes., L’ovocyte de chienne constitue un modèle de méiose très particulier parmi les mammifères. Sa maturation in vitro, étudiée depuis une dizaine d’années seulement, reste très mal maîtrisée : faible taux de métaphase II (10 à 20 % contre plus de 90 % chez les bovins) et fort taux de dégénérescence en culture (20 à 60 %) malgré les essais d’amélioration des milieux de culture. Or la maturation in vitro est une étape indispensable pour avoir accès, tant chez le chien que chez les Canidés en voie de disparition, aux biotechnologies de la reproduction (fécondation et production d’embryons in vitro notamment). Il est indispensable de mieux comprendre la biologie de l’ovocyte chez la chienne pour améliorer les taux de maturation. Nous nous sommes intéressées, en premier lieu, au rôle de l’AMPc dans la reprise de la méiose in vitro. Nous avons modulé la concentration intraovocytaire d’AMPc en soumettant les ovocytes à des molécules qui la diminuent (Rp-AMPc) ou l’augmentent (dbAMPc et forskoline), ou en dénudant l’ovocyte pour arrêter tout apport par les cellules du cumulus. Nous avons ainsi montré que l’AMPc jouerait un rôle dans la poursuite de la méiose dont la reprise serait également contrôlée par une autre voie, peut-être calcique. En parallèle, nous explorons l’évolution de l’ultrastructure de l’ovocyte au cours de la maturation in vivo et in vitro, pour détecter les anomalies cytoplasmiques qui peuvent apparaître au cours de la maturation., Viaris de Lesegno Christine, Tanguy-Dezaux Cécile, Chebrout Martine, Reynaud Karine, Chastant-Maillard Sylvie. Maturation ovocytaire in vitro chez la chienne. In: Bulletin de l'Académie Vétérinaire de France tome 158 n°2, 2005. pp. 173-180.

Canine reproduction has several distinctive features. Firstly, folliculogenesis is unusual as numerous ovarian follicles contain several oocytes (polyovular follicles). Secondly, unlike in other mammalian species, oocytes at the time of ovulation are still at an immature stage (prophase I, germinal vesicle stage), and complete their maturation in the oviduct. This phenomenon is not easy to observe because the canine oocyte has a high lipid content and its DNA is difficult to visualise. Fertilization of immature oocytes has been observed in vitro, however in vivo, fertilization occurs in oocytes at the metaphase II stage, approximately 50 hours after ovulation. The 2-pronuclei stage is reached 72-124 hours after ovulation, and 2-cell embryos are present 96-168 hours after ovulation. The oviductal phase is long and embryos enter the uterine cavity at the morula or early blastocyst stage 10-12 days following ovulation. Implantation occurs 18 to 21 days after ovulation. In spite of all these specificities, studies on canine reproduction were so far mainly clinical. However, current research is focusing on fundamental knowledge, namely the mechanisms controlling oocyte maturation in vivo, in the hope to improve the yield of oocyte maturation in vitro, which is still very low., Plusieurs aspects de la reproduction sont particuliers à l’espèce canine. D’une part, la folliculogenèse est singulière car chez la chienne, de nombreux follicules ovariens contiennent plusieurs ovocytes (follicules poly-ovocytaires). D’autre part, contrairement à ce qui est observé chez les autres femelles de mammifères, au moment de l’ovulation, l’ovocyte est encore à un stade immature (prophase I, stade vésicule germinative ou VG) et la maturation ovocytaire se poursuit ensuite dans l’oviducte. L’observation de ce phénomène est rendue complexe par le fait que l’ovocyte canin est riche en lipides et que son ADN est donc difficile à visualiser. In vitro, la fécondation d’ovocytes immatures a été observée mais in vivo, elle a lieu au moment où les ovocytes ont atteint le stade métaphase II, environ 50 h après l’ovulation. Les premiers pronoyaux sont présents 72 à 124 h après l’ovulation et les premiers embryons au stade 2-cellules sont observés 96 à 168 h après l’ovulation. La période de transit dans l’oviducte est longue et les embryons n’atteignent l’utérus qu’au stade morula ou jeune blastocyste, 10 à 12 jours après l’ovulation et l’implantation a ensuite lieu vers 18 à 21 jours. Globalement, malgré toutes ces particularités, les recherches sur la reproduction dans l’espèce canine étaient jusqu’alors essentiellement cliniques. Les travaux visent maintenant à améliorer les connaissances fondamentales, notamment concernant les mécanismes contrôlant la maturation ovocytaire in vivo, pour pouvoir ensuite améliorer les rendements de la maturation in vitro, actuellement très faibles., Reynaud Karine, Fontbonne Alain, Marseloo Noël, Viaris de Lesegno Christine, Thoumire Sandra, Chastant-Maillard Sylvie. Maturation ovocytaire, fécondation et développement embryonnaire chez la chienne. In: Bulletin de l'Académie Vétérinaire de France tome 158 n°2, 2005. pp. 167-172.

Objective To evaluate the numbers of ovarian follicles during fetal life in the gonads of human female fetuses with the 45,X karyotype (Turner syndrome, TS) and to compare them with those from age-matched 46,XX fetuses. Design Retrospective study. Setting An academic hospital. Patient(s) Ovarian samples from TS fetuses (aged 17–37 weeks), mainly obtained after induced abortion, and ovaries from eight control fetuses (matching gestational ages). Intervention(s) Embedded blocks of ovaries were collected from anatomy-pathology departments of three university hospitals and were sectioned and stained with hematoxylin-eosin. Main outcome measure(s) Observation of primordial and growing follicles. Result(s) In the fetal ovaries of controls, numerous oogonia were observed at 18 weeks. Primordial follicles were present in all ovaries from 20 weeks' gestation onward, whereas preantral and antral follicles were observed from 26 weeks onwards. In ovaries from 45,X TS fetuses, oogonia were observed in some ovaries, but no primordial, preantral, or antral follicles were found, even in ovaries from the third trimester of gestation. Conclusion(s) Follicle formation and growth are severely reduced in ovaries from aborted 45,X TS fetuses.

Resume Le follicule est la structure organisee qui permet la croissance de l’ovocyte et qui amene celui-ci jusqu’a l’ovulation. Dans la majorite des cas, un follicule abrite un seul ovocyte, mais parfois, il peut en contenir plusieurs. Ce type de follicule polyovocytaire est observe chez de tres nombreuses especes de mammiferes et leur frequence est extremement variable d’une espece a l’autre (de

Important reproductive events take place in the canine oviduct in the presence of increasing concentrations of progesterone (P4). To investigate the potential effects of P4 on the canine oviduct, the expression of nuclear (PR) and membrane (PGRMC1 and 2, mPRα, β and γ) P4 receptors was studied by quantitative RT-PCR and immunohistochemistry. Oviducts were collected from Beagle bitches after the onset of pro-oestrus and before the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov) and on Days 1, 4 and 7 post-ovulation (n = 6 bitches/stage). PR mRNA concentrations decreased from Pre-LH to Day 7 in the ampulla and isthmus, whereas both PGRMC1 and 2 mRNA levels increased over the same period. The main change in mPR expression was an increase in mPRβ and γ mRNAs at Day 7 in the isthmus. Furthermore, PR proteins were expressed in the nuclei of luminal epithelial, stromal and muscular cells, whereas the expression of PGRMCs and mPRs was primarily cytoplasmic and localised in the luminal epithelium. The immunostaining for PR decreased at Day 4 in the stroma and muscle, whereas it remained strong in the epithelium from Pre-LH to Day 7. PGRMC1 staining was strong at Days 4 and 7 whereas PGRMC2 was highly expressed from Pre-ov to Day 7. The most intense immunostaining signals for all three mPRs were observed at Day 7. Our results strongly support the hypothesis that P4 is an important regulator of oviductal functions in the bitch through complementary classical and non-classical P4 pathways.

Assay of blood progesterone (P4) is commonly practiced to determine the time of ovulation, diagnose luteal insufficiency, and predict time of parturition in bitches. Because of practical constraints, most blood samples cannot be assayed on site immediately after collection. The aim of this work was to evaluate the effects of various sampling and storage conditions on concentrations of P4 as determined by chemiluminescence immunoassay. The blood of 5 Beagle bitches was collected from the jugular vein to study the effect of the type of collection tube (silicone, lithium heparin, EDTA), the storage time of unseparated or separated plasma (2 h to 14 d), and the number of freeze-thaw cycles (1-10) on P4. The effect of each factor was tested within one assay session. None of the factors significantly affected P4. Thus, P4 appears to remain relatively stable in canine blood samples exposed to various processing and storage conditions.

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–100 nm) membrane-bound vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system in the uterus. However, little is known about the role of these vesicles in the oviduct. Our study aimed at (1) demonstrating the existence of oviducal-embryo communication via exosomes, (2) deciphering the exosomal dialogue between them at the proteomic level, and (3) comparing the exosomal proteomic content to the oviducal fluid proteomic content in order to highlight the key role of exosomes in this dialogue. Cow oviducts (pool of 6 oviducts at different stages of the cycle in 4 replicates) were flushed, and exosomes were isolated by serial ultracentrifugation. Exosomes were measured by dynamic light scattering analysis, resulting in exosomes (63.25–97.03 nm) and microvesicle observations (>100 nm). Bovine embryos were produced in vitro up to the blastocyst and hatching/hatched blastocyst stages. To demonstrate the existence of the oviducal-embryo communication via exosomes, oviducal exosomes were labelled with green fluorescent dye (PKH67), filtered (0.22 µm) to remove microvesicles, and co-incubated with blastocysts and hatching/hatched (H) blastocysts for 20 h, under 5% CO2 and 5% O2 conditions. Subsequently, embryos were washed in exosome-free medium, fixed in 4% paraformaldehyde, and labelled with Hoechst 33342 and Actin Red Phallodin. Confocal microscopy observations confirmed that exosomes were internalized by blastocysts and H-blastocysts and located around the nucleus, demonstrating the existence of an oviducal-embryo communication via exosomes. Moreover, our results showed that the zona pellucida does not represent a barrier for exosomes and they act as natural nanoshuttles bringing oviducal signals into the embryo. Then, proteomic analysis by LC1D-nanoESI-LTQ-Orbitrap was used to decipher oviducal exosomal content, identifying 480 proteins. Gene ontology analysis revealed that a high number of these proteins were involved in metabolism (24.9%), cellular process (19.3%), and 0.8% reproductive processes. Further analysis revealed that more than 56% of exosomal proteins involved in cellular process were associated with cell-to-cell communication. Finally, exosomal proteins were compared with proteins present in oviducal fluid from a pool of samples from cows at Day 0 and Day 10 of the oestrous cycle. Comparative analysis showed that from a total of 607 proteins identified in both oviducal exosomes and fluid sources, 105 were specific to exosomes, 127 were specific to fluid, whereas 375 were common to both sources. Our findings provide the first evidence of oviducal-embryo communication via exosomes, an important first step in furthering the understanding of the oviducal environment and the role of exosomes as early mediators of embryo-maternal cross talk. This research was supported by the EU AgreenSkills fellowship n° 267196 and EU FECUND Project no 312097.

Contents 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 17 beta-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 17 beta-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.

Contents In the bitch, oocyte meiosis resumption takes place in the oviduct. Using oocytes from anestrous bitches, in vitro maturation (IVM) generally gives very poor results. To investigate the contribution of oocyte competence to the low IVM yield, we compared in vivo maturation in an optimal environment with conventional IVM. A total of 418 grade 1 cumulus-oocyte complexes (COCs) from 10 anestrous bitches were transferred into the oviducts of recipient bitches either on Day −1 (n = 3 recipients), Day 0 (n = 2) or on Day +1 (n = 2) relative to ovulation. For each donor bitch, 20 grade 1 COCs were also cultured in vitro. After 72 h of in vivo or IVM, the nuclear stage of oocytes was determined after DNA and tubulin staining. Of the 154 oocytes recovered and examined after intratubal transfer, only 2% reached the metaphase I or II stage and 38.3% were degenerated. Oocytes cultured in vitro displayed a higher metaphase rate (7.6%, n = 170) and lower degeneration rate (12.9%) compared with transferred oocytes (p

Contents In canine species, in vitro maturation (IVM) rates of oocytes collected from anoestrous ovaries are low (

Contents As puppies are born with very low immunoglobulin concentrations, they rely on passive immune transfer from ingested colostrum to acquire a protective immunity during the first few weeks of life. The purpose of this study was to describe the timing of gut closure in canine neonates. Twenty-two Beagle puppies received 3 ml of standardized canine colostrum at 0, 4, 8, 12, 16 or 24 h after birth using a feeding tube. Blood immunoglobulins G (IgG, M and A) were assayed 0, 4 and 48 h after colostrum ingestion. IgG absorption rate was significantly affected by the time of colostrum administration, and the IgG concentrations in puppies serum 48 h after administration were significantly higher when colostrum was ingested at 0–4 h of age than at 8–12 h or 16–24 h (1.68 ± 0.4, 0.79 ± 0.07 and 0.35 ± 0.08 g/l, respectively; p

Reproductive physiology in dogs is quite unusual compared with that in other mammalian species. The peculiarities include the presence of numerous polyoocyte follicles, the ovulation of an immature oocyte (GV stage, non-fertilizable) and a peri-ovulatory period during which concentrations of circulating progesterone are particularly high. The aim of this review is to examine the unusual aspects of the reproductive physiology of dogs and how this relates to the clinical biology of this species.

Syncytins are envelope protein genes of retroviral origin that have been captured for a function in placentation. Two such genes have already been identified in simians, two distinct, unrelated genes have been identified in Muridae, and a fifth gene has been identified in the rabbit. Here, we searched for similar genes in the Laurasiatheria clade, which diverged from Euarchontoglires—primates, rodents, and lagomorphs—shortly after mammalian radiation (100 Mya). In silico search for envelope protein genes with full-coding capacity within the dog and cat genomes identified several candidate genes, with one common to both species that displayed placenta-specific expression, which was revealed by RT-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with precise proviral integration at a site common to dog and cat. Cloning of the gene for an ex vivo pseudotype assay showed fusogenicity on both dog and cat cells. In situ hybridization on placenta sections from both species showed specific expression at the level of the invasive fetal villi within the placental junctional zone, where trophoblast cells fuse into a syncytiotrophoblast layer to form the maternofetal interface. Finally, we show that the gene is conserved among a series of 26 Carnivora representatives, with evidence for purifying selection and conservation of fusogenic activity. The gene is not found in the Pholidota order and, therefore, it was captured before Carnivora radiation, between 60 and 85 Mya. This gene is the oldest syncytin gene identified to date, and it is the first in a new major clade of eutherian mammals.

Contents Over the last 10–15 years, long-acting GnRH agonists have become widely available. In the field of small animal reproduction, most recent studies have focused on the use of two compounds developed under the form of subcutaneous implants: azagly-nafarelin and deslorelin. Only the latter has been commercially available for use in male dogs, first in Australia and New Zealand, then in several countries of the European Union since 2008. Although officially marketed for male dogs, this compound has also been studied in bitches and more recently in queens. Some published papers or recent presentations at congresses – still unpublished – have focused on the use of GnRH agonists implants in females.

Ovarian physiology of prepubertal and adult animals is different. Some characteristics as follicular dynamics (follicular waves and growth) are similar but total follicular population and number of growing follicles are higher in prepubertal ovary. Prepubertal oocytes represent a negative model for in vitro studies since they lead to lower cleavage and blastocyst rates when they are used to produce embryos. This reduced ability to support embryonic development is due to follicular and oocyte differences. Follicular fluid and granulosa cells proteins, and steroidogenic potential differ between prepubertal and adult animals. Moreover, experiments using nuclear transfer demonstrate that cytoplasmic maturation of prepubertal oocytes is incomplete. These deficient oocytes are smaller, contain lower levels of MPF and MAP Kinase and differences in metabolism and cytoplasmic organelles are observed.

Background A large fraction of vertebrate genomes is composed of endogenous retroviruses (ERVs), which are remnants of ancient retroviral infections. In mammals, five retroviral envelope genes with a placenta-specific expression and a fusogenic activity, the syncytins, have been independently “captured” in primates, rodents and lagomorphs (which are all Euarchotonglires mammals) and conserved over more than 30 million years in a functional state. They are involved in the formation of the syncytiotrophoblast, and are necessary for placental development. Here we searched for syncytins in the Carnivora order that belongs to the Laurasiatheria, which diverged from the Euarchotonglires shortly after mammalian radiation around 100 million years ago.

Les oviductes, ou trompes utérines, assurent le transport et la maturation finale des gamètes, abritent la fécondation et les premiers stades de développement embryonnaire. L'environnement tubaire est finement régulé par les stéroïdes ovariens mais aussi par les gamètes et les embryons qui interagissent avec lui. Longtemps considéré comme une simple zone de transit, l'oviducte se voit attribuer aujourd'hui des fonctions plus complexes dans ces différents processus. Le fluide tubaire, de mieux en mieux caractérisé, peut être considéré comme la première interface entre l'organisme maternel et l'embryon et pourrait jouer à ce titre un rôle majeur dans la qualité du conceptus.

The biology of the canine oocyte is unusual compared with that of other mammalian females. The present paper reviews both in vivo and in vitro specificities of canine oocytes. Final follicular growth in the bitch is characterised by an early appearance of LH binding sites in the granulosa, a high proportion of polyovular follicles and a preovulatory luteinisation, starting at the time of the LH surge. Through follicular fluid, preovulatory oocytes are thus exposed to high levels of progesterone, as high as 1000-fold plasma concentrations. The composition of the follicular fluid is affected by the size of the female. The more specific aspect of oocyte biology in the bitch is ovulation: oocytes are expelled immature, at the Prophase I stage. Ovulatory follicles are 6–8 mm in diameter, releasing oocytes from 110 µm, with dark cytoplasm. Resumption of meiosis occurs from 48 h postovulation, MII stages appearing 48–54 h after ovulation. The mechanisms controlling such a late meiotic resumption are still unknown. Granulosa cells seem to play a central role as in other mammalian species, but not with cAMP as the principal mediator. The importance of a transient reactivation of oocyte transcription a few hours before meiotic resumption is to be explored. These specific features may contribute to the low efficiency of IVM. Only 10–20% oocytes reach the metaphase stage and suffer from a poor cytoplasmic maturation. Moreover, in vitro culture of canine oocytes is associated with a high proportion of degeneration. To date, IVM of the oocytes is the main limiting factor for the development of assisted reproductive techniques in the canine. A better knowledge of the basic physiology of folliculogenesis and the molecular mechanisms controlling oocyte meiosis resumption in this species may allow us to overcome this obstacle.

In mammalian ovaries, the vast majority of follicles contain only one oocyte. However, follicles containing several oocytes, or polyovular follicles, are also found in most species. Their frequency ranges from < 0.1% to 14% of the total number of follicles, and they can contain from 2 to 17 oocytes (or even 100 in certain marsupials). Three hypotheses have been proposed to explain their occurrence : division of an oocyte initially containing several nuclei, fusion of several different follicles, or nonseparation of several oocytes at the time of the formation of the primordial follicles. This latter hypothesis seems the most likely. The fate of these follicles has not been studied extensively, and for a long time they were considered as pathological. Studies conducted in pigs and dogs have shown that these polyovular follicles can grow, reach the preovulatory stage, and ovulate. However, all the oocytes in a single follicle are not equal. The examination of oocytes retrieved from a single follicle suggests that only one oocyte shows good morphological characteristics, whereas the others are at various stages of more or less advanced degeneration. The regulation of the number of polyovular follicles is still poorly understood and the data on the effects of age, gonadotropins, and steroids is contradictory. Their frequency can be increased by using treatments with estrogenic effects, or endocrine disruptors. In mice, the control is also genetic. The mechanisms leading to the formation of polyovular follicles remain to be explored, and the study of the functioning of these particular follicles would improve our understanding of the oocyte-follicle dialogue, Dans les ovaires des mammifères, la très grande majorité des follicules ne contient qu’un seul ovocyte. Mais, dans la plupart des espèces, certains follicules contiennent plusieurs ovocytes : on parle alors de follicules polyovocytaires ou multiovocytaires. Leur fréquence varie entre < 0.1 % et 14% des follicules totaux et ils peuvent contenir de 2 à 17 ovocytes (et même plus de 100 chez certains marsupiaux). Trois hypothèses pourraient expliquer leur formation : la division d’un ovocyte ayant initialement plusieurs noyaux, la fusion de plusieurs follicules différents ou la non-séparation de plusieurs ovocytes au moment de la formation des follicules primordiaux. Cette dernière hypothèse semble la plus probable. La destinée de ces follicules a été encore peu explorée et ils ont été longtemps considérés comme pathologiques. Des travaux menés notamment chez le porc et le chien montrent que ces follicules polyovocytaires peuvent grandir, atteindre le stade préovulatoire et ovuler. Cependant, tous les ovocytes d’un même follicule ne sont pas équivalents. L’observation des ovocytes après ponction du follicule suggère qu’un seul ovocyte semble de bonne qualité morphologique, tandis que les autres sont à des stades de dégénérescence plus ou moins avancés. La régulation du nombre des follicules polyovocytaires est peu connue et il existe des données contradictoires sur les effets de l’âge, des gonadotrophines et des stéroïdes. Leur fréquence peut être augmentée par des traitements à effets oestrogéniques ou par l’action de perturbateurs endocriniens. Chez la souris, le contrôle est également génétique. Les mécanismes conduisant à la formation des follicules polyovocytaires restent à explorer et l’étude du fonctionnement de ces follicules pourrait contribuer à une meilleure connaissance du dialogue ovocyte-follicule., Reynaud Karine, Thoumire Sandra, Saint-Dizier Marie, Tahir Zahid, Chebrout Martine, Chastant-Maillard Sylvie. Les follicules ovariens polyovocytaires: physiologiques et pas si rares. In: Bulletin de l'Académie Vétérinaire de France tome 164 n°2, 2011. pp. 127-135.

Background Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation. Methods Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry. Results The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml). Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively. Conclusion Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

International audience; This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n=2), follicular phase (F, n=4), ovulation (O, n=2), early luteal (EL, n=7) and mid/late luteal phase (MLL, n=2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II+III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P

The IGF system plays a crucial role in ovarian folliculogenesis, and changes in IGF-binding protein (IGFBP) levels modulate IGF bioavailability. Data from various mammalian models suggest a link between body size, IGF1 in serum and female reproduction parameters. Among the vertebrate species, the dog exhibits the widest span in body height. Height is known to be positively correlated with the concentration of serum IGF1. In this work, the ovarian physiology of 40 bitches exhibiting a wide span of height, and breed type was investigated. IGF1, IGF2, IGFBP3, estradiol (E2), and progesterone concentrations in plasma and preovulatory follicular fluid were quantified. A total of 455 follicles, 2–8 mm in diameter, were recovered at the preovulatory stage, measured, and punctured. Intrafollicular levels of IGF1 were positively correlated with plasma levels, and plasma IGF1 levels were positively correlated with both bitch height and weight. The concentrations were threefold higher in large dogs compared with small dogs. A positive correlation between intrafollicular and plasmatic IGFBP3 levels and a positive correlation between plasmatic IGFBP3 levels, and both height and weight of the bitches were observed. The number of preovulatory follicles and the diameter of the three largest follicles were positively correlated with bitch height. E2 intrafollicular concentrations were higher in preovulatory follicles from small animals than in those from large animals. In conclusion, the strong variability in height between dogs appeared to be associated with dramatic differences in IGF1, and IGFBP3 levels, in both plasma and follicular fluid. These differences were associated with significant differences in some functional aspects of ovarian follicles.

International audience; Canine embryos are a scarce biological material because of difficulties in collecting in vivo-produced embryos and the inability, to date, to produce canine embryos in vitro. The procedure for the transfer of in vivo-produced embryos has not been developed adequately, with only six attempts reported in the literature that have resulted in the birth of 45 puppies. In vitro, the fertilisation rate is particularly low ( approximately 10%) and the incidence of polyspermy particularly high. So far, no puppy has been obtained from an in vitro-produced embryo. In contrast, cloning of somatic cells has been used successfully over the past 4 years, with the birth of 41 puppies reported in the literature, a yield that is comparable to that for other mammalian species. Over the same period, canine embryonic stem sells and transgenic cloned dogs have been obtained. Thus, the latest reproductive technologies are further advanced than in vitro embryo production. The lack of fundamental studies on the specific features of reproductive physiology and developmental biology in the canine is regrettable in view of the increasing role of dogs in our society and of the current demand for new biological models in biomedical technology.

International audience; In vitro maturation (IVM) of bitch oocytes is, to date, a very inefficient process, with common metaphase rates approximately 0-20% and mean degeneration rates approximately 20-30%. In other mammals, meiotic resumption is controlled in the cumulus-oocyte complex by the disappearance of the coupling between granulosa cells and the oocyte. The first aim of this study was to evaluate the influence on meiotic resumption of a mechanical denudation of the oocytes before maturation. The nuclear stage was determined by DNA staining with ethidium-homodimer-2 under confocal microscopy. Denuded (n = 318) and control (n = 378; no mechanical denudation) oocytes had similar degeneration rates (respectively 32.1 vs 28.6%). However, meiosis resumption rates were significantly higher for denuded oocytes (DO; 26.9 vs 17.8%). Secondly, we aimed to evaluate oocytes experiencing spontaneous denudation during the 72 h IVM period. Denuded oocytes, having lost cumulus cells on at least 75% of their perimeter (n = 440), were compared with surrounded oocytes (SO), with 100% of their perimeter surrounded by granulosa cells (n = 860). As above, the nuclear stage was determined by confocal microscopy, but cytoplasmic maturation was also evaluated through transmission electron microscopy. Degeneration rates but also meiosis resumption and metaphase rates were significantly higher for denuded than for SO (9.6 vs 2.8% for metaphase rate). Nevertheless, ultrastructurally, metaphase DO have scarcer organelles unevenly distributed, with smooth endoplasmic reticulum concentrated in aggregates in the cortical zone. Denudation, whether mechanical or spontaneous, is thus an inefficient mean to obtain metaphase II oocytes suitable for in vitro fertilization.

International audience; Contents This study was designed to describe, both quantitatively (morphometry) and qualitatively (histological differentiation), follicle and oocyte growth in the feline ovary. The ovaries of 43 cats were collected and processed for histology. The diameters of 832 follicle/oocyte pairs were measured, with and without zona pellucida (ZP), and a special emphasis was placed on the study of early folliculogenesis. Primordial, primary, secondary, pre-antral and early antral follicles were measured at 44.3, 86.2, 126.0, 155.6 and 223.8 mum in diameter respectively. A biphasic pattern of follicle and oocyte growth was observed. Before antrum formation, follicle (x) and oocyte (y) size were positively and linearly correlated (y = 0.500x + 20.01, r(2) = 0.89). Antrum formation occurred when the follicle reached 160-200 mum in diameter (when oocyte was at 102 mum). After antrum formation, a decoupling was observed, a minimal increase in oocyte size contrasting with a significant follicle development (y = 0.001x + 114.39, r(2) = 0.01). The pre-ovulatory follicle diameter was approximately 3500 mum and the maximal oocyte diameter was 115 mum. The ZP, absent in primordial and primary follicles, appeared at the secondary stage and reached almost 6 mum at the pre-ovulatory stage. These results suggest that (i) in feline ovary, follicle and oocyte growth pattern is similar to that observed in other mammals; (ii) the antrum forms in 160-200 mum follicles, which represents 5% of the pre-ovulatory diameter and (iii) the oocyte had achieved more than 90% of its maximal growth at the stage of antrum formation.

In most mammals, oocytes are ovulated at the metaphase II stage, and the meiosis inhibition is then lifted by fertilization. In bitches and other Canidae species however, oocytes are released at the prophase I stage, and another 48 to 72h are necessary for them to mature into the metaphase II stage and become fertilizable. This specificity is currently hindering the development of reproductive biotechnologies in these species. In vitro maturation rates of canine oocytes are very low, as only 10 to 30% will reach the metaphase stage after 72h in culture. In bitches, nuclear maturation occurs in the oviduct, and tubal derivatives (culture media, such as Synthetic Oviductal Fluid, oviductal explants, coculture on tubal cell layers) were used to improve the yield, but so far not very successfully. This failure may be due to the lack of data on the composition of oviductal fluid in bitches. Further studies on the oviductal microenvironment of bitches are therefore necessary, as it is probably quite different from the oviductalmicroenvironment of other females, e. g. the presence of preovulatory luteinisation in bitches only. Creating a maturation medium based on the composition of oviductal fluid could be an interesting avenue to explore to improve in vitro maturation rates., Chez la plupart des mammifères, les ovocytes sont bloqués en métaphase II au moment de l’ovulation et cette inhibition de la méiose est ensuite levée par la fécondation. Chez les chiennes et les autres femelles de canidés, les ovocytes sont libérés au stade de prophase I, et il faut encore attendre 48 à 72 heures pour qu’ils atteignent le stade de métaphase II et deviennent fécondables. Cette particularité constitue aujourd’hui un frein au développement des biotechnologies de la reproduction chez les canidés. En effet, dans les essais de maturation in vitro d’ovocytes canins, seuls 10 à 30% des ovocytes atteignent le stade de métaphase au bout de 72 heures de culture. Chez la chienne, la maturation nucléaire se produisant dans l’oviducte, des substituts de l’oviducte (milieux de culture comme le Synthetic Oviductal Fluid, explants d’oviductes, cultures sur tapis de cellules tubaires) ont été utilisés pour les cultures d’ovocytes in vitro, afin d’en améliorer le rendement, mais sans grand succès jusqu’à maintenant. Cet échec peut être dû au manque de données sur la composition du liquide tubaire de la chienne. L’étude de ce microenvironnement prend donc tout son intérêt, celui-ci étant probablement assez différent de celui des autres femelles, ne serait-ce que par l’existence du processus de lutéinisation préovulatoire dans cette espèce. À terme, la conception d’un milieu de maturation sur la base de la composition du liquide tubaire pourrait être une voie intéressante pour augmenter les taux de maturation in vitro., Fontaine Emmanuel, Reynaud Karine, Thoumire Sandra, Chebrout Martine, Faure Laetitia, Segalini Vincent, Chastant-Maillard Sylvie. Microenvironnement tubaire: rôle dans la maturation des ovocytes canins in vivo et in vitro. In: Bulletin de l'Académie Vétérinaire de France tome 162 n°2, 2009. pp. 145-154.

International audience; Cumulus-oocyte complexes (COCs) were recovered from ovaries of bitches during anoestrus. The ultrastructural organisation of COCs was determined before and after 72 h in vitro maturation (IVM) by transmission electron microscopy. The aim of the study was to determine the quality of oocytes used for IVM and to assess cytoplasmic maturation of IVM metaphase (M) II oocytes. In addition, we examined whether the oocytes that did not reach MII were engaged in an erratic maturation process or whether they were blocked during their progression through a normal maturation process. Before IVM, there were two populations of oocytes: (1) oocytes with a centrally located germinal vesicle, a transcriptionally active aspect and an immature cytoplasm; and (2) oocytes with an eccentric nucleus, a transcriptionally inactive aspect and a more mature cytoplasm. After IVM, most oocytes were still at the germinal vesicle stage with three different patterns and all showing a good synchronisation between nuclear and cytoplasmic maturation. MI oocytes had a similar cytoplasmic maturation to that observed in vivo, but failed to complete meiosis; however, IVM MII oocytes had a very poor cytoplasmic maturation. Ultrastructural analysis demonstrated that even when nuclear maturation is achieved, cytoplasmic maturation may not be obtained in vitro. Thus, all IVM systems should be evaluated on both criteria.

International audience; This study was designed to describe the follicular population present on the canine ovary (Canis familiaris) during the preovulatory period and essentially the changes in oocyte size, mucification, and chromatin configuration occurring from before the luteinizing hormone (LH) surge up to postovulation. In a first experiment, ovaries of beagle bitches were collected before (n=21) or after LH surge but before ovulation (post-LH surge/preovulation stage, n=24) as determined using hormone (LH, estradiol, progesterone) assays and ultrasonography. All large (>2mm) follicles were measured and punctured. The numbers of oocytes collected per follicle and the degree of cumulus mucification were recorded. In a second experiment, ovaries were similarly collected before (n=13) and after the LH surge but before ovulation (n=11) as well as after ovulation as determined by ultrasonography (n=9). Chromatin configuration of the oocytes was observed by DNA staining and confocal microscopy. In Experiment 1, before the LH peak, an average of 13.5+/-0.7 follicles per bitch (total 284 follicles) were detected, and the maximal follicle diameter reached 6.5mm. Large follicles were observed already in this period of the cycle and as early as when progesterone was still below 0.5 ng/mL. After the LH peak but before ovulation, 11.0+/-0.7 follicles were present (total 264 follicles). Fully mucified cumulus cells were observed only in follicles larger than 4mm. Multi-oocytic follicles represented 7% (before LH peak) and 4% (after LH peak) of the follicular population. In Experiment 2, all the oocytes were at the germinal vesicle (GV) stage, but three chromatin configurations could be distinguished: diffuse, partly grouped, and fully grouped chromatin. The proportion of oocytes with fully grouped chromatin increased with the follicular diameter and the time in estrus, the maximum being observed after the LH peak. These results suggest that (1) before LH peak, follicles are already of large diameter, similar to the ones at ovulation; (2) the ability for cumulus mucification is acquired during the late steps of follicular growth; (3) three GV patterns may be observed during the periovulatory period.

Article online in advance of print; International audience; The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).

There is very little data available on the specificities of oocyte and embryo biology in bitches. The main difference with other mammals is the time of meiosis resumption : it does not occur before ovulation, but in the oviduct 48 to 60 hours later. The factors responsible for this delay are not known, which may explain why current in vitro maturation rates are so low (10 to 30%). Oocyte harvesting is also a major limiting factor, as there is no effective protocol for the induction of cycles and superovulation. In vitro fertilisation rates are equally low (10%), with a high rate of polyspermia. No puppy has yet been born from an embryo produced in vitro. As for embryos produced in vivo, their collection is difficult due to anatomical reasons and to the fact that superovulation cannot be induced. Embryo transfer to donor bitches is also hindered by difficulties to synchronise ovulations between donor and recipient bitches. Only 6 such trials have been reported in the literature, resulting in the birth of 45 puppies. In vitro cultures are very rarely used, and only four puppies were born from somatic cell cloning with only few hours of in vitro culture. Canine reproductive biotechnologies have thus largely fallen behind, due to a lack of fundamental research to improve our understanding of its specific physiological mechanisms. This deficit is all the more damaging that dogs are increasingly used as relevant biomedical models., La biologie de l’ovocyte et de l’embryon canins, qui présentent des particularités spécifiques, est très mal connue. La chienne se distingue principalement par les modalités de reprise de la méiose ovocytaire : celle-ci n’a pas lieu au moment de l’ovulation, comme chez les autres femelles mammifères, mais dans l’oviducte 48 à 60 heures après l’ovulation. Les facteurs responsables de ce retard ne sont pas connus. Ceci explique sans doute pourquoi les taux de maturation in vitro obtenus à l’heure actuelle sont faibles (10 à 30 %). La collecte des ovocytes est aussi un facteur limitant majeur, en l’absence de protocole efficace d’induction des cycles et de superovulation. Les rendements de fécondation in vitro sont également faibles (10 %), avec un taux élevé de polyspermie. À l’heure actuelle, aucun chiot n’est encore né à partir d’un embryon produit in vitro. Quant aux embryons produits in vivo, leur collecte est difficile pour des raisons anatomiques et le rendement est limité par l’impossibilité d’induire des superovulations ; par ailleurs, leur transfert chez des femelles receveuses se heurte aux difficultés de synchronisation des ovulations entre la femelle donneuse et les receveuses, et la littérature ne décrit que six essais, qui ont abouti à la naissance de 45 chiots. Avec un très faible recours à la culture in vitro, quatre naissances de chiots ont été obtenues par clonage de cellules somatiques. Les biotechnologies de la reproduction sont donc largement en retard dans l’espèce canine, qui souffre d’un manque de travaux fondamentaux visant à mieux comprendre ses mécanismes physiologiques spécifiques. Ce déficit est d’autant plus dommageable que le chien prend une place croissante et pertinente en tant que modèle biomédical., Chastant-Maillard Sylvie, Saint-Dizier Marie, Viaris de Lesegno Christine, Chebrout Martine, Thoumire Sandra, Reynaud Karine, Fontbonne Alain. Biotechnologies de l'embryon dans l'espèce canine. In: Bulletin de l'Académie Vétérinaire de France tome 160 n°2, 2007. pp. 153-161.

International audience; A key limiting step in fertility is the search for the oocyte by spermatozoa. Initially, there are tens of millions of sperm cells, but a single one will make it to the oocyte. This may be one of the most severe selection processes designed by evolution, whose role is yet to be understood. Why such a huge redundancy is required and what does that mean for the search process? we discuss here these questions and consequently new lines of interdisciplinary research needed to find possible answers.