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This work was carried out to compare the endocrine function of ovarian tissue isolated in vitro, in an identical environment between ewes and ewe lambs. Furthermore, to determine whether the differences in endocrine and reproductive function of ewes and ewe lambs are related to differences in the proteomics of corpora lutea, follicles and oocytes. Oestradiol concentrations in tissue cultured in TCM-199 were similar for ewe and ewe lamb follicles collected post slaughter on day 9 to 12 of the oestrous cycle and cultured for different incubation times but increased with increase in follicular size. Oestradiol secretion was greater (P<0.001) for ewe and ewe lamb follicles cultured in media with FCS. Media progesterone concentrations were higher (P<0.001) for ewe than ewe lamb follicles. Progesterone in media and in follicular fluid was increased with increased follicles size. Ewe CL, collected on day 9 to 12 of the oestrous cycle, produced more progesterone than ewe lamb CL when cultured in TCM-199 with or without FCS, PVA, BSA. Proteomics indicated more large spots, in ewe follicular and CL tissue average gels compared with ewe lamb average gels. The protein spots were estimated to be between 45 to 97 kD, in both tissue and age groups, this range of molecular weight could have affected steroid hormone synthesis. (Chapter.3). Ewe and ewe lamb follicles cultured with FSH and LH produced more oestradiol than without, furthermore, oestradiol concentration increased with follicle size. There was no difference in media oestradiol concentration between age groups after 24 h of culture. However, for follicles cultured for 2, 4 or 6 h, concentrations were greater after 4 and 6 h, in ewes than in ewe lambs. Overall ewe lamb follicles produced more progesterone (P<0.001) than ewe follicles when cultured with FSH and LH when cultured for 24 h, but no difference was observed after 2, 4 and 6 hours between ewes and ewe lambs Overall ewe follicles produced more (P<0.002) progesterone than ewe lambs when cultured with different concentration of hCG although there was no difference between ages with respect to oestradiol concentrations. Ewe CL secreted more progesterone (P<0.002) than ewe lamb CL, when cultured for 0 or 24 h. Furthermore, tissue concentrations were greater in ewe CL than ewe lamb CL after incubation in TCM-199, TCM-199 plus BSA, TCM-199 plus FCS and TCM-199 plus PVA. Ewe lamb CL produced more progesterone than ewe CL in medium containing LH when cultured for 2, 4 and 6 hours, but ewes produced more progesterone than lambs when CL were cultured with different concentrations of hCG. Relative to untreated shells, the protein profiles of the ewe follicular shells treated with FSH and LH changed to a greater extent than that of the ewe lambs treated gels in both ages contained more protein spots than control gels. The largest spots were estimated to be between 30 and 97 kDa (Chapter.4). There was no difference between age groups for follicles from ewes and ewe lambs treated with ovagen in oestradiol and progesterone concentrations observed after 2,4, 6 and 8 h of incubation in TCM-199. However, treatment with ovagen plus hCG resulted in higher oestradiol and progesterone concentrations in the media from ewe follicles compared to ewe lambs. Furthermore, there were more protein spots in the range 30 to 66 kDa marker in gels from ewes treated with either ovagen or ovagen plus hCG than for ewe lambs (Chapter 5). Lamb oocytes were smaller than ewe oocytes and developed to a lesser extent in culture. Furthermore, the addition of FCS to TCM-199 caused greater cytoplasmic and nuclear maturation than other media used in this experiment and ewe lamb oocytes have a similar 1D protein bands compared with ewe oocytes, but contained less protein (Chapter 6).

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Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

L’implantation est caractérisée par les premiers contacts cellulaires permanents entre l’endomètre, tapissant l’utérus, et le conceptus (disque embryonnaire et tissus extra-embryonnaires). Cette étape se trouve être l’un des plus importants points de contrôle de la gestation nécessitant un dialogue finement régulé entre ces deux entités. Concernant les ruminants, un déclin de la fertilité a été observé notamment chez les vaches laitières hautes productrices. La moitié des gestations s’arrête pendant la période pré-implantatoire due à des mortalités embryonnaires précoces ainsi qu’à des défauts utérins. Depuis 10 ans, des analyses exploratoires ont été mises en place dans le but d’étudier les profils d’expression de gènes endométriaux sous l’influence du cycle oestral, de la gestation précoce ou encore des stéroïdes ovariens comme la progestérone et les oestrogènes. Ces études sont essentielles pour l’identification des gènes endométriaux clés pour la survie et la croissance du conceptus avant l’implantation. Notre laboratoire a réalisé une analyse transcriptomique à partir d’échantillons endométriaux collectés sur des vaches cycliques et gestantes au 20 ème jour post-oestrus correspondant respectivement à la phase folliculaire et au premier jour d’implantation. Plusieurs familles de facteurs de transcription apparaissent différentiellement exprimées dans cette étude, notamment FOXL2, un membre de la famille des Forkhead Box transcription factor considéré comme le gène clé de la différenciation ovarienne. Ce travail de thèse s’est intéressé à l’implication de FOXL2 dans la physiologie endométriale. FOXL2 est exprimé et régulé pendant le cycle oestral et la gestation précoce dans l’endomètre de ruminants. De plus, la progestérone a été identifiée comme le régulateur majeur de l’expression endométriale de FOXL2 chez la vache et la brebis alors que l’effet des estrogènes n’a pas été démontré. A partir d’une approche gènes candidats, la surexpression de FOXL2 induit la régulation différentielle de onze gènes potentiellement cibles de FOXL2 dans des cultures primaires endométriales de cellules stromales et épithéliales glandulaires. En particulier, PTGS2 qui est un gène impliqué dans la réceptivité utérine apparait inhibé par FOXL2 alors que SCARA5 et RSAD2, tout deux impliqués dans la réponse immunitaire sont stimulés. Enfin, DLX5 apparait différentiellement régulé entre les cellules stromales et épithéliales glandulaires sous l’impact d’une surexpression de FOXL2. Pour conclure, l’expression endométriale de FOXL2 est fortement liée au processus de réceptivité utérine qui se déroule avant l’implantation et peut moduler l’expression de gènes endométriaux essentiels. De nouvelles analyses sont nécessaires pour déterminer si FOXL2 est le gardien de la physiologie reproductive femelle. / Implantation is characterized by the first permanent cellular interactions between the endometrium, lining the uterus, and the conceptus (embryonic disk and extra-embryonic tissues) and appears to be one of the most important checkpoints of successful pregnancy. Regarding ruminant species, and more specifically dairy cows, half of pregnancies abort during the pre-implantation period due to early embryonic death and uterine defects. In the last decade, exploratory approaches have been developed to study endometrial genes expression under the influence of oestrous cycle, early pregnancy, and ovarian steroid hormones in order to identify systematically crucial endometrial genes for conceptus growth and survival leading to a successful implantation in ruminant specifically. A microarray analyse made at the laboratory based on endometrial samples collected from cyclic and pregnant cows at 20 days post-oestrous, corresponding respectively to the follicular phase and the implantation initiation. Several members of the transcription factor families appeared to be differentially expressed in this study including FOXL2, a member of the Forkhead box L sub-class originally considered as a key gene for ovarian differentiation. My PhD thesis focused on the implication of FOXL2 gene in endometrial physiology. FOXL2 gene had been demonstrated to be expressed and regulated during the oestrous cycle and early pregnancy in ruminant endometrium. Moreover, progesterone was identified as a master regulator of FOXL2 endometrial expression in both cattle and sheep whereas estrogens have no impact. Based on candidate genes approach, over-expression of FOXL2 gene induces a regulation of eleven putative FOXL2 target genes in primary endometrial stromal and glandular epithelial cells. In particular, PTGS2 which is a positive regulator gene for uterine receptivity was shown to be inhibited whereas SCARA5 and RSAD2 expressions that were involved in immune response were shown to be stimulated as well as DLX5 expression was differentially regulated between stromal and glandular epithelial cells. Collectively, FOXL2 endometrial expression is strongly linked to the uterine receptivity process prior to the implantation and modulates the expression of essential endometrial genes. Further investigations will be required to investigate whether FOXL2 is the gatekeeper of female reproduction in the vertebrate species.

Prostaglandins are integral mediators in reproductive processes but their exact role in uterine function is still not clear. In addition, ethical restraints have limited the availability of human tissue to investigate uterine prostanoid receptor populations. The aim of this thesis was to characterise the prostanoid receptors on the human and rat myometrium in order to evaluate the potential of the rat as an animal model of human uterine function and disease. For functional analysis of myometrial prostanoid receptors the immersion technique was utilised. LC-ESI-MS/MS was also used to measure the ex vivo myometrial release of prostanoid metabolites. The results show that both the rat and human uterus displays cyclical changes in uterine motility, with myogenicity greatest in the follicular and oestrus stages. The data also indicate that whilst the human uterus is responsive to EP3, EP2, TP, FP and IP receptor agonists, a functional population of only EP3, EP2 and FP receptors is present on the rat uterus, although the TP receptor appears to be upregulated at gestation and post-partum. The results also show that myometrial prostanoid release in the human uterus is cyclically regulated, with the greatest amount of prostaglandins being released during the late follicular stage. In conclusion, although similarities do exist with regard to the ovarian regulation of uterine motility in both the rat and human uterus, the differences in the apparent functional prostaglandin receptor populations between the two species suggest further work is required before the rat can be used as a model of human uterine function.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.