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O objetivo do experimento I foi avaliar a luteólise causada por três doses de PGF2&#945; (12,5; 25 e 50 mg de Dinoprost tometamina) nos dias 5 e 7 do ciclo estral. Foram utilizadas 339 vacas não lactantes da raça Nelore. Os animais foram divididos em dois grupos de acordo com a apresentação do estro, recebendo a dose de PGF2&#945; nos dias 5 e 7 do ciclo estral. Cada grupo foi subdividido em três, que receberam os seguintes tratamentos de PGF2&#945; (12,5 mg; 25 mg; 50 mg). Através da concentração de P4 foram estimadas as taxas de regressão luteal 1 e 0,5 (1 - animais com concentração de P4 abaixo de 1 ng/mL; 0,5 animais com concentração de P4 abaixo de 0,5 ng/mL). Não houve interação entre a dose de PGF2&#945; e o dia do ciclo estral. A aplicação de PGF2&#945; no dia 7 do ciclo estral apresentou maior taxa de regressão luteal 1 e 0,5 quando comparada com o dia 5 do ciclo estral (1 - 76,9 vs 37,0 % - P = 0,0001; 0,5 - 57,5 vs 21,7 %, P = 0,0001, respectivamente). A taxa de regressão luteal 1 aumentou de acordo com a dose de PGF2&#945; administrada (12,5 mg 39,0 %; 25 mg 56,9 %; 50 mg 76,5 % P <0,0001). Quando a PGF2&#945; foi administrada no dia 5 do ciclo estral a concentração média de P4 segui o padrão de luteólise parcial e foi dependente da dose de PGF2&#945;. Quando a PGF2&#945; foi aplicada no dia 7 do ciclo estral a concentração média de P4 caiu drasticamente de 0 para 24 h e não voltou a se elevar em 48 h. A concentração de P4 em 48 h após a aplicação de PGF2&#945; foi menor na dose de 50 mg (0,51 ± 0,07 ng/mL). A taxa de luteólise foi menor no dia 5 do ciclo estral comparado com o dia 7 do ciclo estral. À medida que a dose de PGF2&#945; foi aumentada, a porcentagem de regressão luteal se elevou. O objetivo do experimento II foi avaliar se a substituição das aplicações do GnRH por BE, ECP e eCG no protocolos de 5 dias de P4 causa dupla ovulação e se a utilização de 1 ou 2 mg de BE no início do protocolo influencia na taxa de ovulação dupla. Foram utilizadas 85 multíparas da raça Nelore. No dia 0 do protocolo, as vacas receberam um implante de P4 e a dose de BE segundo o tratamento pertencente (tratamento A 1 mg de BE, tratamento B 2 mg de BE). Cinco dias após, o dispositivo foi retirado e aplicou-se PGF2&#945;, ECP eCG. Houve 5,9 % de dupla ovulação. O tratamento A causou menor porcentagem folículos maiores que 8 mm no dia 7 protocolo que o tratamento B (28,7 vs 50,6 P = 0,0460). Não houve diferença significativa na taxa de ovulação dupla, no diâmetro do folículo dominante no dia 5 e no dia 7 do protocolo e na taxa de crescimento folicular entre os tratamentos A e B. O protocolo de 5 dias de P4 com BE, ECP e eCG causou uma baixa taxa de dupla ovulação. O objetivo do experimento III foi comparar o aparecimento e a frequência de estro e a taxas de ovulação e gestação entre os protocolos de 5 dias de P4 + GnRH / GnRH e de 7 dias de P4 + BE / ECP + eCG em nulíparas, primíparas e multíparas. Foram utilizadas 411 fêmeas da raça Nelore (nulíparas - n = 198; primíparas - n = 80; multíparas - n = 133). No protocolo de 7 dias de P4, os animais receberam no dia 0 um implante de P4 e BE. No dia 7, o dispositivo foi retirado e aplicou-se PGF2&#945;, ECP e eCG. No protocolo de 5 dias de P4, os animais receberam no dia 0 o implante de P4 e GnRH. No dia 5, o dispositivo foi retirado e aplicaram-se 2 doses de PGF2&#945; com intervalo de 6 h entre as doses de PGF2. Os animais que não apresentaram estro até a hora da IA receberam 100 &#956;g de GnRH no momento da IA. A taxa de prenhez utilizando o protocolo de 5 ou 7 dias de P4 variou de acordo com a categoria da fêmea (nulíparas - 41,0 vs 51,0 % - P = 0.1608; primíparas 25,6 vs 31,7 % - P = 0,5513; multíparas - 58,4 vs 32,8 % - P = 0,0041, respectivamente). A taxa de apresentação de estro no protocolo de 7 dias de P4 foi maior para todas as categorias de fêmeas quando comparado com o protocolo de 5 dias deP4. (nulíparas 95,8 vs 66,0 % - P <0,0001; primíparas 48,7 vs 0 %; multíparas - 76,9 vs 13,4 % - P <0,0001, respectivamente). A resposta ao protocolo de 5 dias com GnRH foi pior nas multíparas. / The objective of the experiment I was to evaluate the luteolysis caused by three doses of PGF2&#945; (12.5, 25 and 50 mg of Dinoprost tometamina) when applied on the 5th and 7th days of the estrous cycle. Three hundred thirty-nine (339) nonlactating Nelore cows were used. The animals were divided into two groups according to the onset of estrus, and received the PGF2&#945; dose on the 5th or 7th day of the estrous cycle. Each group was divided into three subgroups, which were submitted to the treatments of PGF2&#945; with dose of 12.5 mg, 25 mg or 50 mg. Through the P4 concentration, the rates of 1 and 0.5 luteal regression were estimated (1 - animals with P4 concentration below 1 ng/mL and 0.5 - animals with P4 concentration below 0.5 ng/mL). There was no interaction between the PGF2&#945; dose and the day of the estrous cycle. The PGF2&#945; application on the 7th day of the estrous cycle had higher rates of the 1 and 0.5 luteal regression, when compared to the PGF2&#945; application on the 5th day of the estrous cycle (1 - 76.9 vs 37.0 % - P = 0.0001; 0,5 - 57.5 vs 21.7 %, P = 0.0001, respectively). The rate of 1 luteal regression increased with the PGF2&#945; dose (12.5 mg - 39.0 %; 25 mg - 56.9 %; 50 mg - 76.5 %, P < 0.0001). The average concentrations of P4, when the PGF2&#945; was administered on the 5th day of the estrous cycle, follow the partial luteolysis standard that dependent on the PGF2&#945; dose. When the PGF2&#945; is applied on 7th day of the estrous cycle, the average concentration of P4 drops dramatically from 0 to 24 h and it do not rise again in 48 h. The P4 concentration is lower in the 50 mg (0.51 ± 0.07 ng/mL), 48 h after the PGF2&#945; application. The luteolysis rate was low on the 5th day of the estrous cycle. The luteal regression percentage increased with increase of the PGF2&#945; dose. The objective of the experiment II was to evaluate whether the replacement of the GnRH applications by BE, ECP and eCG in the 5-day protocols of P4 cause double ovulation and if the use of 1 or 2 mg of BE at the beginning of the protocol influences the double ovulation rate. Eighty-five (85) multiparous Nelore cows were used. On day 0 of the protocol, the cows received a P4 implant and a dose of BE that depending on the treatment to which the cows belong (Treatment A - 1 mg of BE, treatment B - 2 mg of BE). Five days later, the device was removed and the PGF2&#945;, ECP and eCG were applied. In the experiment II, there was 6.5 % of double ovulation. There was no significant difference in the double ovulation rate, dominant follicle diameter on the 5th and 7th day of the protocols, and follicular growth rate between the treatments A and B. The 5-day protocol of P4 with BE, eCG and ECP caused a low rate of the double ovulation, and there was no difference between 1 or 2 mg of BE to synchronize the follicular development wave. The objective of the experiment III was to compare the onset and frequency of estrus and the ovulation and pregnancy rates among the protocols of 5 days of P4 with GnRH and 7 days of P4 with BE, ECP and eCG in nulliparous, primiparous and multiparous. Four hundred eleven (411) Nellore females were used (nulliparous - n = 198; primiparous - n = 80; multiparous - n = 133). In 7-day protocol of P4, the animals received an implant of P4 and BE on day 0. On day 7, the device was removed and the PGF2&#945;, ECP and eCG were applied in the cows. In 5-day protocol of P4, the animals received implants of GnRH and P4 on the day 0. On day 5, the device was removed and two doses of PGF2&#945; were applied with interval of 6 h. The animals that did not show estrus until the AI time received 100 mg of GnRH at this moment. The pregnancy rate varied according to the female category in both protocols (nulliparous - 41.0 vs 51.0 % - P = 0.1608; primiparous - 25.6 vs 31.7 % - P = 0.5513; multiparous - 58.4 vs 32.8 % - P = 0.0041, respectively). The rate estrus onset in the 5-day protocol of P4 with GnRH was lower for all female categories, when compared to the 7-day protocol (nulliparous - 95.8 vs 66.0 % - P <0.0001; primiparous 0 vs 48.7 %; multiparous - 76.9 vs 13.4 % - P <0.0001, respectively). The response in the 5-day protocol with GnRH was worse in multiparous cows.

O objetivo do experimento I foi avaliar a luteólise causada por três doses de PGF2&#945; (12,5; 25 e 50 mg de Dinoprost tometamina) nos dias 5 e 7 do ciclo estral. Foram utilizadas 339 vacas não lactantes da raça Nelore. Os animais foram divididos em dois grupos de acordo com a apresentação do estro, recebendo a dose de PGF2&#945; nos dias 5 e 7 do ciclo estral. Cada grupo foi subdividido em três, que receberam os seguintes tratamentos de PGF2&#945; (12,5 mg; 25 mg; 50 mg). Através da concentração de P4 foram estimadas as taxas de regressão luteal 1 e 0,5 (1 - animais com concentração de P4 abaixo de 1 ng/mL; 0,5 animais com concentração de P4 abaixo de 0,5 ng/mL). Não houve interação entre a dose de PGF2&#945; e o dia do ciclo estral. A aplicação de PGF2&#945; no dia 7 do ciclo estral apresentou maior taxa de regressão luteal 1 e 0,5 quando comparada com o dia 5 do ciclo estral (1 - 76,9 vs 37,0 % - P = 0,0001; 0,5 - 57,5 vs 21,7 %, P = 0,0001, respectivamente). A taxa de regressão luteal 1 aumentou de acordo com a dose de PGF2&#945; administrada (12,5 mg 39,0 %; 25 mg 56,9 %; 50 mg 76,5 % P <0,0001). Quando a PGF2&#945; foi administrada no dia 5 do ciclo estral a concentração média de P4 segui o padrão de luteólise parcial e foi dependente da dose de PGF2&#945;. Quando a PGF2&#945; foi aplicada no dia 7 do ciclo estral a concentração média de P4 caiu drasticamente de 0 para 24 h e não voltou a se elevar em 48 h. A concentração de P4 em 48 h após a aplicação de PGF2&#945; foi menor na dose de 50 mg (0,51 ± 0,07 ng/mL). A taxa de luteólise foi menor no dia 5 do ciclo estral comparado com o dia 7 do ciclo estral. À medida que a dose de PGF2&#945; foi aumentada, a porcentagem de regressão luteal se elevou. O objetivo do experimento II foi avaliar se a substituição das aplicações do GnRH por BE, ECP e eCG no protocolos de 5 dias de P4 causa dupla ovulação e se a utilização de 1 ou 2 mg de BE no início do protocolo influencia na taxa de ovulação dupla. Foram utilizadas 85 multíparas da raça Nelore. No dia 0 do protocolo, as vacas receberam um implante de P4 e a dose de BE segundo o tratamento pertencente (tratamento A 1 mg de BE, tratamento B 2 mg de BE). Cinco dias após, o dispositivo foi retirado e aplicou-se PGF2&#945;, ECP eCG. Houve 5,9 % de dupla ovulação. O tratamento A causou menor porcentagem folículos maiores que 8 mm no dia 7 protocolo que o tratamento B (28,7 vs 50,6 P = 0,0460). Não houve diferença significativa na taxa de ovulação dupla, no diâmetro do folículo dominante no dia 5 e no dia 7 do protocolo e na taxa de crescimento folicular entre os tratamentos A e B. O protocolo de 5 dias de P4 com BE, ECP e eCG causou uma baixa taxa de dupla ovulação. O objetivo do experimento III foi comparar o aparecimento e a frequência de estro e a taxas de ovulação e gestação entre os protocolos de 5 dias de P4 + GnRH / GnRH e de 7 dias de P4 + BE / ECP + eCG em nulíparas, primíparas e multíparas. Foram utilizadas 411 fêmeas da raça Nelore (nulíparas - n = 198; primíparas - n = 80; multíparas - n = 133). No protocolo de 7 dias de P4, os animais receberam no dia 0 um implante de P4 e BE. No dia 7, o dispositivo foi retirado e aplicou-se PGF2&#945;, ECP e eCG. No protocolo de 5 dias de P4, os animais receberam no dia 0 o implante de P4 e GnRH. No dia 5, o dispositivo foi retirado e aplicaram-se 2 doses de PGF2&#945; com intervalo de 6 h entre as doses de PGF2. Os animais que não apresentaram estro até a hora da IA receberam 100 &#956;g de GnRH no momento da IA. A taxa de prenhez utilizando o protocolo de 5 ou 7 dias de P4 variou de acordo com a categoria da fêmea (nulíparas - 41,0 vs 51,0 % - P = 0.1608; primíparas 25,6 vs 31,7 % - P = 0,5513; multíparas - 58,4 vs 32,8 % - P = 0,0041, respectivamente). A taxa de apresentação de estro no protocolo de 7 dias de P4 foi maior para todas as categorias de fêmeas quando comparado com o protocolo de 5 dias deP4. (nulíparas 95,8 vs 66,0 % - P <0,0001; primíparas 48,7 vs 0 %; multíparas - 76,9 vs 13,4 % - P <0,0001, respectivamente). A resposta ao protocolo de 5 dias com GnRH foi pior nas multíparas. / The objective of the experiment I was to evaluate the luteolysis caused by three doses of PGF2&#945; (12.5, 25 and 50 mg of Dinoprost tometamina) when applied on the 5th and 7th days of the estrous cycle. Three hundred thirty-nine (339) nonlactating Nelore cows were used. The animals were divided into two groups according to the onset of estrus, and received the PGF2&#945; dose on the 5th or 7th day of the estrous cycle. Each group was divided into three subgroups, which were submitted to the treatments of PGF2&#945; with dose of 12.5 mg, 25 mg or 50 mg. Through the P4 concentration, the rates of 1 and 0.5 luteal regression were estimated (1 - animals with P4 concentration below 1 ng/mL and 0.5 - animals with P4 concentration below 0.5 ng/mL). There was no interaction between the PGF2&#945; dose and the day of the estrous cycle. The PGF2&#945; application on the 7th day of the estrous cycle had higher rates of the 1 and 0.5 luteal regression, when compared to the PGF2&#945; application on the 5th day of the estrous cycle (1 - 76.9 vs 37.0 % - P = 0.0001; 0,5 - 57.5 vs 21.7 %, P = 0.0001, respectively). The rate of 1 luteal regression increased with the PGF2&#945; dose (12.5 mg - 39.0 %; 25 mg - 56.9 %; 50 mg - 76.5 %, P < 0.0001). The average concentrations of P4, when the PGF2&#945; was administered on the 5th day of the estrous cycle, follow the partial luteolysis standard that dependent on the PGF2&#945; dose. When the PGF2&#945; is applied on 7th day of the estrous cycle, the average concentration of P4 drops dramatically from 0 to 24 h and it do not rise again in 48 h. The P4 concentration is lower in the 50 mg (0.51 ± 0.07 ng/mL), 48 h after the PGF2&#945; application. The luteolysis rate was low on the 5th day of the estrous cycle. The luteal regression percentage increased with increase of the PGF2&#945; dose. The objective of the experiment II was to evaluate whether the replacement of the GnRH applications by BE, ECP and eCG in the 5-day protocols of P4 cause double ovulation and if the use of 1 or 2 mg of BE at the beginning of the protocol influences the double ovulation rate. Eighty-five (85) multiparous Nelore cows were used. On day 0 of the protocol, the cows received a P4 implant and a dose of BE that depending on the treatment to which the cows belong (Treatment A - 1 mg of BE, treatment B - 2 mg of BE). Five days later, the device was removed and the PGF2&#945;, ECP and eCG were applied. In the experiment II, there was 6.5 % of double ovulation. There was no significant difference in the double ovulation rate, dominant follicle diameter on the 5th and 7th day of the protocols, and follicular growth rate between the treatments A and B. The 5-day protocol of P4 with BE, eCG and ECP caused a low rate of the double ovulation, and there was no difference between 1 or 2 mg of BE to synchronize the follicular development wave. The objective of the experiment III was to compare the onset and frequency of estrus and the ovulation and pregnancy rates among the protocols of 5 days of P4 with GnRH and 7 days of P4 with BE, ECP and eCG in nulliparous, primiparous and multiparous. Four hundred eleven (411) Nellore females were used (nulliparous - n = 198; primiparous - n = 80; multiparous - n = 133). In 7-day protocol of P4, the animals received an implant of P4 and BE on day 0. On day 7, the device was removed and the PGF2&#945;, ECP and eCG were applied in the cows. In 5-day protocol of P4, the animals received implants of GnRH and P4 on the day 0. On day 5, the device was removed and two doses of PGF2&#945; were applied with interval of 6 h. The animals that did not show estrus until the AI time received 100 mg of GnRH at this moment. The pregnancy rate varied according to the female category in both protocols (nulliparous - 41.0 vs 51.0 % - P = 0.1608; primiparous - 25.6 vs 31.7 % - P = 0.5513; multiparous - 58.4 vs 32.8 % - P = 0.0041, respectively). The rate estrus onset in the 5-day protocol of P4 with GnRH was lower for all female categories, when compared to the 7-day protocol (nulliparous - 95.8 vs 66.0 % - P <0.0001; primiparous 0 vs 48.7 %; multiparous - 76.9 vs 13.4 % - P <0.0001, respectively). The response in the 5-day protocol with GnRH was worse in multiparous cows.

The studies presented in this thesis were designed to elucidate whether the abundance of ovulation-inducing factor/nerve growth factor (OIF/NGF) in alpaca semen can be used as a biomarker to predict male fertility. The neurotrophin, OIF/NGF has been identified in camelid, cattle and human semen. It is only in camelids, however, that an ovulation-inducing role for OIF/NGF has been described. The information gathered from several studies clearly demonstrate that this protein is the stimulus responsible for initiating the ovulatory cascade in camelids. In addition, intramuscular administration of OIF/NGF resulted in a dose-dependent response in terms of ovulation rate, corpus luteum (CL) lifespan, luteinizing hormone (LH) and progesterone secretion. I hypothesized that the quantity of OIF/NGF differs among male alpacas and this abundance arbitrates ovulation and pregnancy rates as well as CL formation and function. To substantiate this hypothesis, the following questions were answered: 1) can OIF/NGF in alpaca semen be quantified using a radioimmunoassay; 2) does the concentration and total abundance of OIF/NGF in alpaca semen vary within and among male ejaculates; 3) what is the glandular source of OIF/NGF that contributes to the male ejaculate; 4) is OIF/NGF concentration or abundance related to parameters associated with male fertility; 5) can OIF/NGF concentration or total abundance in the ejaculate discriminate fertile and subfertile males using both retrospective and prospective approaches; and 6) can power Doppler ultrasonography be used to assess the luteotrophic effect of OIF/NGF in tissue vasculature of the developing CL? I discovered that the source and the amount of OIF/NGF varies among species. In llamas, OIF/NGF is produced by both the corpus and disseminate portions of the prostate gland. In rats, OIF/NGF was detected in testis interstitial cells and in the lumen of the coagulating gland (anterior prostate). Ovulation-inducing factor/NGF secretion by the ampullae and vesicular glands contributed to its presence in bull (cattle and bison) ejaculates. In elk and white tail deer, OIF/NGF was detected in the ampullae and prostate glands, respectively. To gain an understanding of the abundance of OIF/NGF in ejaculates and changes in its concentration within and among males, OIF/NGF levels in semen were quantified using the radioimmunoassay. The assay developed exhibited parallel displacement curves among recombinant NGF, OIF/NGF purified from llama seminal plasma, llama and bull (cattle) seminal plasma. Ovulation-inducing factor/NGF comprised a greater percentage of the total protein found in camelid ejaculates than in cattle. Ovulation-inducing factor/NGF concentration correlated positively with sperm concentration and negatively with pH and semen volume, while total abundance of OIF/NGF was related to total prostate area and OIF/NGF concentration. Although a correlation was found between sperm concentration, neither OIF/NGF concentration nor total abundance was associated with higher ovulation, pregnancy or live birth rates. A clear association of the quantity of OIF/NGF in the male ejaculate at breeding and CL form and function was not evident. The measurement of CL vasculature by power Doppler ultrasonography, however, was able to determine nonpregnancy in alpacas earlier than the assessment of changes in CL diameter. In summary, my results did not support the hypothesis that the measurement of OIF/NGF concentration or total abundance in alpaca semen can be used to predict fertility in male alpacas.

La saisonnalité de la reproduction consiste en l’alternance entre des périodes d’activité et de repos sexuel. Ce phénomène s’observe chez les caprins de la région subtropicale du Mexique et rend impossible la reproduction à contre saison. La réintroduction d’un mâle dans un groupe de femelles peut rompre l’anoestrus saisonnier et rapidement stimuler leur activité ovarienne. Cependant, l’utilisation de boucs induits à un comportement sexuel intense pendant la période de repos sexuel au moyen d’un traitement lumineux –mâles photo-stimulés- est nécessaire afin d’assurer la qualité de la stimulation fournie par ceux-ci. Le temps de contact entre les sexes est un autre facteur pouvant moduler la réponse des femelles et il a longtemps été suggéré que ce contact devait être maintenu de manière continue (24h/24) pendant plusieurs jours afin que la majorité des femelles ovulent. L’objectif de cette thèse a été de déterminer si une réduction du temps de contact journalier entre les sexes diminue les réponses ovulatoire et reproductrice de chèvres en anoestrus saisonnier exposées aux mâles. / Seasonality of reproduction consists of alternations between periods of sexual activity and sexual rest (anoestrus). This seasonality is observed in goats from subtropical Mexico and makes impossible out-of-season reproduction. The reintroduction of a male in a group of females can break the seasonal anoestrus and quickly stimulate ovarian activity. However, the use of bucks induced to a high sexual activity during the sexual rest by a treatment of long days –photo-stimulated males- is necessary to ensure the quality of stimulation provided by them. The duration of contact between sexes is another factor that modulates the response of females and it has long been suggested that it should be maintained continuously (24h/24) for several days so that the majority of females ovulate. The purpose of this thesis was to determine whether a reduction of daily contact between sexes decrease ovulatory and reproductive responses of goats exposed to males during the seasonal anoestrus. The results indicate that duration of contact between sexes can be drastically reduced from 16 to 1 h per day without reducing the ovulatory and reproductive responses of females.

Chez les rongeurs, les facteurs sociaux sont connus pour pouvoir moduler la transition pubertaire. Ainsi une jeune souris femelle mise en contact avec un mâle adulte présentera une ouverture vaginale plus précoce qu’une femelle isolée du mâle. L’objectif de ma thèse a été de caractériser les conséquences d’une exposition précoce au mâle dans l’espèce caprine. Les caprins sont une espèce dont la reproduction est saisonnée et permettant, de par sa taille, une étude plus fine des sécrétions endocrines que les rongeurs. Nos résultats ont permis de mettre en évidence que la présence de boucs sexuellement actifs induit une puberté précoce chez les chevrettes, l’ovulation pouvant être induite dès l’âge de 3 mois et demi. Les femelles présentent suite à cette première ovulation une cyclicité régulière ainsi qu’une maturation précoce du tractus génital. Le niveau d’activité sexuelle du bouc est un facteur crucial à l’induction d’une puberté précoce chez la chèvre puisque la présence de mâles castrés n’a aucun effet et que les femelles sont toutes pubères dans le mois suivant l’entrée en saison sexuelle des mâles. Ce travail démontre, dans l’espèce caprine, un rôle crucial de l’environnement social dans la régulation de la maturation sexuelle. Plus particulièrement, cela met en évidence que la présence de boucs peut réactiver efficacement et de manière très précoce l’axe gonadotrope de jeunes chèvres immatures. / In rodents, social factors are known to modulate the pubertal transition. Hence, young female mice exposed to adult male exhibit an earlier vaginal opening than young females isolated from male. The aim of my thesis was to characterize the consequences of a precocious exposure to male in another specie, goats. Goats are seasonal breeders and due to their size the fine study of endocrine secretions is easier than in rodents. Our results highlighted that an early exposure to sexually active bucks induces a precocious puberty in young female goats. The first ovulation can be induced as early as 3.5 months old, following this induced first ovulation, goats remain cycling regularly. Females precociously exposed to bucks also exhibit an acceleration of the genital tract maturation. The level of sexual activity of the male is a crucial criteria to induce a precocious puberty in goats as exposure to castrated bucks had no effect on the age at puberty. Moreover, all females exposed to intact bucks ovulated for the first time within a month after buck started to exhibit sexual behaviors. This work revealed, in goats, a crucial role of the social environment on the regulation of sexual maturation. More precisely, it highlights that exposure to bucks is highly efficient to reactivate precociously the gonadotrope axis of youg immature goats.

Le récepteur nucléaire Nr5a2 est exprimé dans l’ovaire, plus spécifiquement dans les cellules de granulosa et lutéales. Une déplétion conditionnelle de Nr5a2 dans les cellules de granulosa au stade de follicule primaire par croisement de souris Nr5a2-flox et Amhr2-Cre (Nr5a2f/fAmhr2Cre/+) génère des problèmes au niveau de l’expansion du cumulus, de l’ovulation et de la lutéinisation. Ainsi, nous estimons que Nr5a2 régule les connexions intercellulaires dans le follicule ovarien via la connexine 43 (Cx43), une protéine de jonction impliquée dans l’expansion du cumulus. Le premier objectif de l’étude était de déterminer si l’absence d’expansion du cumulus chez les souris Amhr2Cre-cKO est liée à l’absence de communication intercellulaire adéquate entre les cellules de granulosa et de cumulus dans les follicules préovulatoires. À cette fin, des ovaires de souris immatures Amhr2Cre-cKO et non transgéniques ont été prélevés (n=3) après un traitement de superstimulation utilisant les gonadotropines eCG suivie de hCG afin d’induire l’ovulation. Nous avons ainsi démontré, par RT-PCR, une sous-expression de Cx43 avant et au moment du stimulus ovulatoire (0 h et 2 h) chez le groupe Amhr2Cre-cKO (P<0.01), ce qui pourrait mener à un problème dans l’acquisition de la compétence développementale de l’oocyte. D’un autre côté, au moment de l’ovulation (12 h), l’ARNm de Cx43 est surexprimé dans le groupe Amhr2Cre-cKO, ce qui pourrait prévenir les cellules du cumulus de se détacher l’une de l’autre. Nous avons ainsi conclu que Cx43 est un gène sous le contrôle de Nr5a2 et qu’une régulation erronée de ce gène est une cause possible du problème d’expansion du cumulus chez les souris Amhr2Cre-cKO. Afin d’examiner le rôle de Nr5a2 dans l’ovulation et la lutéinisation à différents stades de la maturation folliculaire, nous suggérons que Nr5a2 module la séquence temporelle des événements menant à l’ovulation. En croisant des souris Nr5a2-flox et Cyp19-Cre (Nr5a2f/fCyp19Cre/+), l’expression de Nr5a2 a été interrompue dans les cellules de granulosa des follicules antraux et préovulatoires. Aucune portée n’a été obtenue de ces souris (n=4) durant un essai d’accouplement de 6 mois. Chez les souris Cyp19Cre-cKO on remarque la présence de structures s’apparentant à des cellules de type lutéales et les femelles âgées d’un an présentent des kystes folliculaires hémorragiques et une hypertrophie de l’épithélium en surface de l’ovaire. Les deux modèles transgéniques démontrent donc une absence de l’expansion du cumulus et de l’ovulation. En conclusion, Nr5a2 semble réguler différemment la folliculogenèse et l’ovulation dans les cellules de granulosa des follicules primaires et antraux. / The nuclear receptor Nr5a2 is expressed in the ovary, exclusively in granulosa and luteal cells. Conditional disruption of Nr5a2 in granulosa cells beginning with primary follicles by means of Nr5a2-floxed and Amhr2-Cre mice (Nr5a2f/fAmhr2Cre/+) results in failure in cumulus expansion, ovulation and luteinization. We hypothesize that Nr5a2 regulates intercellular connections in ovarian follicles through connexin 43 (Cx43), a gap-junctional protein related to cumulus expansion. The first objective of this study was to determine whether the lack of cumulus expansion in Amhr2Cre-cKO mice is related to the absence of normal cell-to-cell communication in cumulus/granulosa cells of preovulatory follicles. To address this, immature ovaries of Amhr2Cre-cKO and non-transgenic littermates mice were collected (n=3) after superstimulation to induce follicle development and ovulation. Using RT-PCR, the Cx43 mRNA levels were shown to be downregulated prior to and at the time of the ovulatory stimulus (0 h and 2 h) in the Amhr2Cre-cKO group (P<0.01), which may lead to the failure in the acquisition of oocyte developmental competence. On the other hand, by the time of ovulation (12 h), mRNA levels of Cx43 are upregulated in Amhr2Cre-cKO group, which may prevent the cumulus cells to detach one from another. We conclude that Cx43 is one of the downstream genes under Nr5a2 control and its dysregulation can be one reason for the defect in cumulus expansion in Amhr2Cre-cKO females. To examine the role of Nr5a2 in ovulation and luteinization in different stages of the follicle maturation, we hypothesized that Nr5a2 modulates the events leading to ovulation in a temporal sequence. By crossing Nr5a2-floxed and Cyp19-Cre mice (Nr5a2f/fCyp19Cre/+), Nr5a2 was disrupted in granulosa cells of antral and preovulatory follicles. No litters were born to Cyp19Cre-cKO females (n=4) during a 6 months breeding trial. Cyp19Cre-cKO enabled the development of a luteal-like structure, and 1-year-old females presented hemorrhagic follicular cysts and hypertrophic ovarian surface epithelium. Both knockout models display lack of cumulus expansion and ovulation. We conclude that Nr5a2 differentially regulates folliculogenesis and ovulation in granulosa cells of small and antral follicles.

Le récepteur nucléaire Nr5a2 est exprimé dans l’ovaire, plus spécifiquement dans les cellules de granulosa et lutéales. Une déplétion conditionnelle de Nr5a2 dans les cellules de granulosa au stade de follicule primaire par croisement de souris Nr5a2-flox et Amhr2-Cre (Nr5a2f/fAmhr2Cre/+) génère des problèmes au niveau de l’expansion du cumulus, de l’ovulation et de la lutéinisation. Ainsi, nous estimons que Nr5a2 régule les connexions intercellulaires dans le follicule ovarien via la connexine 43 (Cx43), une protéine de jonction impliquée dans l’expansion du cumulus. Le premier objectif de l’étude était de déterminer si l’absence d’expansion du cumulus chez les souris Amhr2Cre-cKO est liée à l’absence de communication intercellulaire adéquate entre les cellules de granulosa et de cumulus dans les follicules préovulatoires. À cette fin, des ovaires de souris immatures Amhr2Cre-cKO et non transgéniques ont été prélevés (n=3) après un traitement de superstimulation utilisant les gonadotropines eCG suivie de hCG afin d’induire l’ovulation. Nous avons ainsi démontré, par RT-PCR, une sous-expression de Cx43 avant et au moment du stimulus ovulatoire (0 h et 2 h) chez le groupe Amhr2Cre-cKO (P<0.01), ce qui pourrait mener à un problème dans l’acquisition de la compétence développementale de l’oocyte. D’un autre côté, au moment de l’ovulation (12 h), l’ARNm de Cx43 est surexprimé dans le groupe Amhr2Cre-cKO, ce qui pourrait prévenir les cellules du cumulus de se détacher l’une de l’autre. Nous avons ainsi conclu que Cx43 est un gène sous le contrôle de Nr5a2 et qu’une régulation erronée de ce gène est une cause possible du problème d’expansion du cumulus chez les souris Amhr2Cre-cKO. Afin d’examiner le rôle de Nr5a2 dans l’ovulation et la lutéinisation à différents stades de la maturation folliculaire, nous suggérons que Nr5a2 module la séquence temporelle des événements menant à l’ovulation. En croisant des souris Nr5a2-flox et Cyp19-Cre (Nr5a2f/fCyp19Cre/+), l’expression de Nr5a2 a été interrompue dans les cellules de granulosa des follicules antraux et préovulatoires. Aucune portée n’a été obtenue de ces souris (n=4) durant un essai d’accouplement de 6 mois. Chez les souris Cyp19Cre-cKO on remarque la présence de structures s’apparentant à des cellules de type lutéales et les femelles âgées d’un an présentent des kystes folliculaires hémorragiques et une hypertrophie de l’épithélium en surface de l’ovaire. Les deux modèles transgéniques démontrent donc une absence de l’expansion du cumulus et de l’ovulation. En conclusion, Nr5a2 semble réguler différemment la folliculogenèse et l’ovulation dans les cellules de granulosa des follicules primaires et antraux. / The nuclear receptor Nr5a2 is expressed in the ovary, exclusively in granulosa and luteal cells. Conditional disruption of Nr5a2 in granulosa cells beginning with primary follicles by means of Nr5a2-floxed and Amhr2-Cre mice (Nr5a2f/fAmhr2Cre/+) results in failure in cumulus expansion, ovulation and luteinization. We hypothesize that Nr5a2 regulates intercellular connections in ovarian follicles through connexin 43 (Cx43), a gap-junctional protein related to cumulus expansion. The first objective of this study was to determine whether the lack of cumulus expansion in Amhr2Cre-cKO mice is related to the absence of normal cell-to-cell communication in cumulus/granulosa cells of preovulatory follicles. To address this, immature ovaries of Amhr2Cre-cKO and non-transgenic littermates mice were collected (n=3) after superstimulation to induce follicle development and ovulation. Using RT-PCR, the Cx43 mRNA levels were shown to be downregulated prior to and at the time of the ovulatory stimulus (0 h and 2 h) in the Amhr2Cre-cKO group (P<0.01), which may lead to the failure in the acquisition of oocyte developmental competence. On the other hand, by the time of ovulation (12 h), mRNA levels of Cx43 are upregulated in Amhr2Cre-cKO group, which may prevent the cumulus cells to detach one from another. We conclude that Cx43 is one of the downstream genes under Nr5a2 control and its dysregulation can be one reason for the defect in cumulus expansion in Amhr2Cre-cKO females. To examine the role of Nr5a2 in ovulation and luteinization in different stages of the follicle maturation, we hypothesized that Nr5a2 modulates the events leading to ovulation in a temporal sequence. By crossing Nr5a2-floxed and Cyp19-Cre mice (Nr5a2f/fCyp19Cre/+), Nr5a2 was disrupted in granulosa cells of antral and preovulatory follicles. No litters were born to Cyp19Cre-cKO females (n=4) during a 6 months breeding trial. Cyp19Cre-cKO enabled the development of a luteal-like structure, and 1-year-old females presented hemorrhagic follicular cysts and hypertrophic ovarian surface epithelium. Both knockout models display lack of cumulus expansion and ovulation. We conclude that Nr5a2 differentially regulates folliculogenesis and ovulation in granulosa cells of small and antral follicles.

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel. / Identification of regulatory elements in the genome is of paramount importance to understanding the mechanisms governing the expression of specific genes in a given cell type. Following the LH surge, the ovarian peri-ovulatory follicle enters an intensive program of cellular differentiation, orchestrated by major changes in the transcriptional profile of granulosa cells, ultimately triggering ovulation and luteinization, processes essentials for fertility in females. In the mouse, several genes essential to the success of this program are induced 2 to 6 hours after the ovulatory stimulus. Using a standard protocol for superovulation in mice, the regulatory elements were isolated and identified in granulosa cells 4h after administration of hCG using the method FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combined with next generation sequencing. The results of this analysis demonstrate that after the ovulatory stimulus, granulosa cells undergo a major reprogramming of regulatory elements, which is correlated with the extensive changes in their biological functions. In addition, this study showed that most regulatory elements were associated with distal intergenic regions and introns, indicating that these regions are important in transcriptional regulation in granulosa cells. A variety of transcriptional regulators known to be essential for ovarian function, and their binding sites were also identified in this analysis, demonstrating that the FAIRE method has the power to predict molecular events that have correlates in the known physiology of ovarian processes.

Comprendre les événements paracriniens qui régulent la fertilité chez la vache est nécessaire non seulement en raison de l'importance agricole de cette espèce, mais aussi pour son utilisation potentielle comme modèle chez l’humain. L'oxyde nitrique (NO), un gaz de radicaux libres, a été impliqué dans la croissance folliculaire et l'ovulation chez les rongeurs et d'autres espèces, mais chez la vache c’est une énigme fascinante : le NO est produit par les cellules de la granulosa bovine et est régulé par la FSH, mais la présence et le profil d'expression des enzymes responsables de la synthèse de NO (NOS) dans les cellules de la granulosa tout au long de la croissance folliculaire ne sont pas claires. Les objectifs de cette thèse sont: (1) élucider le mécanisme de contrôle des NOS et les conséquences de la production d'oxyde nitrique pour le fonctionnement des cellules de la granulosa au cours du développement folliculaire chez la vache et (2) déterminer la régulation des NOS pendant la cascade ovulatoire induite par LH chez les cellules de la granulosa bovine et si l'activité des NOS pour l’expression des gènes critiques dans la cascade ovulatoire chez cette espèce. Les résultats sont séparés en 2 articles. Dans le premier article, la régulation de NOS2 dans les cellules de la granulosa bovine a été explorée. L'abondance des ARNm codant pour NOS2 a été stimulée par la FSH et l’IGF1 en augmentant l’estradiol, et un blocage de l'action de l’estradiol a conséquemment réduit les niveaux d'ARNm codant pour NOS2. De plus, l'inhibition de l'activité des NOS a augmenté l'apoptose dans les cellules de la granulosa in vitro. Dans le second article, il a été démontré que le pic de LH induit une activation des NOS dans les cellules de la granulosa, et que l'activité de NOS induit la production de NO, ce qui est essentiel pour l’expression des gènes critiques dans la cascade ovulatoire induite par LH comme EREG/AREG/PTGS2. Ensemble, les résultats présentés dans ces 2 articles suggèrent que les niveaux physiologiques d'activité des NOS peuvent contribuer à la croissance et la survie des cellules de la granulosa et indiquent également que NO peut être essentiel pour l'ovulation chez les bovins. / Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth and ovulation in rodents and other species, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the presence and the expression pattern in granulosa cells of the enzymes responsible for NO synthesis (NOS) throughout follicular growth are unclear. The objectives of the present thesis were (1) to elucidate the mechanism of control of NOS and the consequences of nitric oxide production for granulosa cell function during follicle development in cattle; and (2) to determine the regulation of NOS during the LH-induced ovulatory cascade in bovine granulosa cells and whether NOS activity is critical for the ovulatory cascade in this species. The results are separated in 2 articles. In the first article, the regulation of NOS2 in bovine granulosa cells was explored. Abundance of mRNA encoding NOS2 was stimulated by FSH and IGF1 through increased estradiol, and a blockade of estradiol action consequently lowered NOS2 mRNA levels. Further, inhibition of NOS activity increased apoptosis in granulosa cells in vitro. In the second article, it was demonstrated that the LH surge induces NOS activation in granulosa cells, and that NOS activity induces the production of NO, which is essential for EREG/AREG/PTGS2 expression, critical genes in the LH-induced ovulatory cascade. Together, the results presented in these 2 articles suggest that physiological levels of NOS activity may contribute to growth and survival of granulosa cells, and also indicate that NO may be essential for ovulation in cattle.