This study was designed to investigate the locus of action of different gonadotrophins on the stimulation of multiple follicular development and superovulation in heifers. In addition follicular populations and steroiodogenic potential of the follicles (>10 mm diameter) at different stages of the superovulatory treatment were studied. Experiment 1 was carried out to investigate the effect of three gonadotrophins (pregnant mare serum gonadotrophin, PMSG; human menopausal gonadotrophin, hMG-Pergonal 75 i.u. FSH, 75 i.u. LH per ampoule; and urofollitrophin UF-Metrodin 75 i.u. FSH per ampoule) on multiple follicular development and hormonal profiles. Experiments 2 and 3 were designed to study follicular populations and steroiodogenic potential of the follicles in superovulated heifers and to compare these parameters with control heifers.

Proper understanding of ovarian follicular and luteal development is essential to improve and optimally control reproductive function in domestic animals and to unravel causes of infertility in animals and humans. MicroRNAs (miRNAs) are key post-transcriptional gene regulators in multiple processes involving tissue growth and differentiation. The studies in this thesis were carried out to identify and characterise expression profiles of miRNAs and their potential roles during ovarian follicular development in the cow. The first study aimed to identify expression profiles of miRNAs during antral follicle growth. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. An heterologous microarray approach followed by RT-qPCR validation was used to identify and compare miRNA profiles between large (13–16 mm) healthy and small (4–8 mm) follicles. A unique subset of 7 miRNAs (miR-144, miR-202, miR-210, miR-451, miR-652, miR-873 and miR-876) was identified that were enriched in the Large Healthy follicles relative to small follicles and Large Atretic follicles. Further characterisation revealed that many of these miRNAs were highly expressed in the granulosa compartment of the follicle, predominantly in the mural granulosa cells, indicating their potential involvement in regulating granulosa cell function. Expression patterns of miRNAs in the ovarian tissues were generally reflected in the follicular fluid, thus follicular fluid may be used to monitor follicular health. Finally, tissue-wide screening of miRNAs identified miR-202 as a gonad-specific miRNA in the cow. The aim of the second study was to identify potential roles of the miRNAs enriched in Large Healthy follicles. Using in silico approaches, about 1700 bovine genes were identified as the predicted targets of those miRNAs. Putatively targeted signalling pathways are primarily involved in cell survival, proliferation and differentiation. Further, as expected, top predicted gene targets (SPRED1, ATG7, TGFBR2) showed an expression pattern in follicular tissues that was opposite to the patterns of the targeting miRNAs. However, expression patterns of miR-202 or miR-210 during follicular growth could not be recapitulated in gonadotrophin-stimulated cells in vitro and moreover, over-expression or inhibition of miR-202 in these cells did not result in changes in target mRNA levels. The third study investigated the expression profiles of miRNAs during follicle-luteal transition. Microarray analyses identified 9 miRNAs that were upregulated and 14 miRNAs that were downregulated between Large Healthy follicles and early corpus luteum in the cow. Predicted targets of these miRNAs were associated with signalling pathways involved in cell survival, proliferation and differentiation, indicating that these miRNAs might play significant regulatory roles during ovulation and early luteal development. Further, expression of some of these miRNAs and their putative targets during luteinisation in vivo could be recapitulated in cultured granulosa cells providing a system to study the functional roles of these miRNAs during follicle-luteal development. In summary, this is the first study identifying unique subsets of miRNAs associated with follicular development and the follicle-luteal transition in the cow which may be important for female fertility.

The overall aim of this thesis was to determine if day old turkey gonads could be cryopreserved and transplanted into recipient poults. This would allow for grafts to develop and mature normally and potentially produce donor-derived offspring. In addition, the monitoring of folliculogenesis in chickens was studied to determine if ultrasonography would be a useful technique to study this biological process, with the intention of using this method in future studies on ovarian graft development. Three studies were conducted: cell and tissue viability of vitrified day old turkey gonads, transplantation of day old turkey gonads into recipient poults, and monitoring of follicle growth in chickens using ultrasonography. The objective of the first study was to determine if day old turkey gonads were viable after vitrification using a standard protocol or with variation in equilibrium solution (ES) and/or vitrification solution (VS) absorption times. Three different ES time points were tested 10, 15 and 20 min (10ES, 15ES and 20ES) and two different VS time points 2 and 3 min (2VS and 3 VS). The cell and tissue viability was determined by Trypan Blue Assay and light microscopy, respectively. Testicular cell viability was conducted using three vitrification protocols and fresh tissue. All vitrification protocols along with fresh tissue were assessed by light microscopy, to evaluate histological alterations, in ovarian and testicular tissue. Protocols with the highest cell viability and best morphological scores were selected as being the most suitable for cryopreservation. Testicular tissue vitrified using 15ES or 20ES with 3VS had the highest cell viability. Ovarian and testicular tissue vitrified using 15ES with (3VS or 2VS), showed the best morphological scores, out of all the protocols. The second study was broken down into two parts: Part A (Trial 1 & 2) was to determine the most suitable age group for poults pre-surgery to give the highest survivability; Part B (Trial 3) was to determine if previously vitrified day old turkey gonads could develop and mature normally, by retrieving grafts post-surgery, at- different time points. In Trial 1, large white turkeys (LWT) 1, 3, 4 and 7 days of age were used and for Trial 2, LWT’s aged 1, 3, 4, and 5 days of age were used. In Trial 3, bronze turkeys at 1 day of age were used, and graft tissue was used from day old LWT’s previously vitrified (10ES/2VS) or fresh. For all Trials, the survivability at each time point was analyzed, and for the third Trial, the grafts recovered were histologically analysed. From Trials 1 and 2, seven and three day old poults had the highest survivability ratios (3/5 and 6/8) respectively. For Trial 3, day old male poults (96%) had a higher survivability than the females (68%). From Trial 3, transplants were only recovered in females and males up until 4 days and 4 weeks post-surgery respectively, with no fresh tissue grafts recovered. The histological analysis of testicular transplants showed deteriorating structure, with a steady progression away from normal morphology, post-surgery. The third study’s objective was to determine the growth rates and patterns of folliculogenesis in Barred Plymouth Rock (BPR) hens by using ultrasonography. Two ultrasound Trials were performed: the first to determine the optimum time interval between serial ultrasound scans to accurately map follicles, and the second to tackle the main objective of the third part of this thesis. For the first Trial, BPR hens were scanned periodically over 24 hours, follicle diameter and position were recorded and mapped with respect to the ovary and neighbouring follicles. Proportional follicle growth, compared to the first scanning session showed that the 24hr time point had the only significant (P<0.001) proportional follicle growth. It is recommended here that scans occur twice a day (morning and afternoon) to capture a more precise growth rate of follicles. In the second Trial, BPR hens were scanned twice a day, over an 11-day period. Follicle diameters (height and width) were recorded to calculate follicle area. The growth of each follicle’s area was compared to the time before ovulation to determine the overall follicle growth rate. Additionally, it was determined if time (night, day) and type of preovulatory follicle (F1-F5) played a significant role in follicle growth rates. The overall follicle growth rate was best described by a cubic equation (R2=0.907, P<0.001). Follicle growth rates were influenced by both time (P=0.009) and type (P<0.001). With F2 and F3 follicles (P<0.05) having a higher growth rate than F1 and F5 types. In conclusion, modifications to the standard vitrification protocol used on quail gonads were necessary to increase cell viability and lower morphological alterations for turkey gonads left whole. For future work it has been shown that day old turkey poults can survive gonad transplantation. The lack of development of grafts is most likely due to a combination of tissue damage after vitrification-warming procedures and insufficient immunosuppression of the host. This work has paved the way for the poultry industry to be able to cryogenically store turkey gonads and revive lines when required. Additionally it was shown that serial ultrasound scans twice a day provided accurate monitoring of follicle growth in Barred Plymouth Rock hens. For BPR hen’s follicle growth rates and patterns were successfully measured. This gives the industry another tool to better select superior laying hens and to create a more homogeneous laying flock. Future application of ultrasonography on gonad monitoring has the potential to show growth and maturation of grafted tissue before the production of donor-derived offspring, enabling earlier detection of successful transplantation.

Made available in DSpace on 2014-06-11T19:30:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-27Bitstream added on 2014-06-13T20:39:56Z : No. of bitstreams: 1 amorim_jpa_me_botib.pdf: 5424546 bytes, checksum: 5ed4b7c62a36ad76439e56113f8aabc3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nas fêmeas dos Teleostei a produção ilimitada de oócitos deve-se à constante proliferação das oogônias situadas no epitélio germinativo das lamelas ovígeras. Ao entrarem em meiose dão origem aos oócitos que são envoltos por uma camada de células foliculares, formando os folículos. No interior dos folículos, os oócitos passam por diferentes estágios de desenvolvimento (crescimento primário e secundário/vitelogênese, e maturação) ao final dos quais estão prontos para a desova. Os folículos pós-ovulatórios regridem e os óocitos que não tiveram sucesso na ovulação entram em atresia. A compreensão de como ocorre a foliculogênese nos Teleostei é recente e as descrições existentes referem-se aos grupos mais derivados. Por outro lado, os diferentes estágios de desenvolvimento dos oócitos, numa nova visão, vêm sendo utilizados na descrição de diferentes classes reprodutivas, ao longo do processo de maturação gonadal que ocorre a cada ano. Para testar a aplicabilidade desses novos conceitos aos Teleostei mais basais, analisou-se a foliculogênese e procedeu-se a caracterização celular das classes reprodutivas em Pimelodus maculatus, através de parâmetros histológicos, ultraestruturais e imunocitoquímico. Em P. maculatus a proliferação mitótica das oogônias, ocorre sempre acima da membrana basal, dá origem a conjuntos de células que são gradual e individualmente envoltos pelas células epiteliais, formando um tipo de cisto. As oogônias no interior dos cistos, ou oogônias secundárias entram em meiose ou dividem novamente 1, 2, 3 ou mais vezes por mitose e entram em meiose dando origem aos ovócitos. O conjunto de células, oócitos envoltos pelas células originárias do epitélio, forma uma espécie de sáculo, que se projeta para o estroma, mas permanece conectado ao próprio epitélio, compartilhando a mesma... / The unlimited production of oocytes in Teleostei females is due to the constant proliferation of the oogonia located in the germinal epithelium of ovigerous lamellae. Upon entering meiosis, oogonia give origin to the oocytes, which are surrounded by a layer of follicular cells and thus form the follicles. Within the follicles, oocytes undergo different developmental stages (primary and secondary growth/vitellogenesis, and maturation) followed by spawning. Post-ovulatory follicles regress and the oocytes that failed to ovulate enter atresia. The understanding of how folliculogenesis occurs in Teleostei has been recently achieved, and the descriptions available to date refer to the most derived groups. On the other hand, oocyte developmental stages have been used, under this new view, to describe the different reproductive classes seen along the gonadal maturation process that takes place every year. In order to test the applicability of these new concepts to the most basal Teleostei, this study aimed at analyzing folliculogenesis, and characterizing the cells found in these reproductive classes in Pimelodus maculatus, by using histological, ultrastructural and immunocitochemical parameters. In P. Maculatus, oogonia mitotic proliferation always occurs above the basement membrane, giving rise to cell sets which are gradually and individually surrounded by epithelial cells forming a sort of cyst. The oogonia located within the cysts, or secondary oogonia, either enter meiosis or divide again 1, 2, 3 or more times through mitosis, giving origin to oocyte. The cell set, oocytes surrounded by the cells originated from the epithelium, forms a sac-like structure that projects into the stroma, but remains connected to the epithelium sharing the same basement membrane. The early meiotic cells, which are small, have a basophilic nucleus, and a clear and scarce cytoplasm... (Complete abstract click electronic access below)

Folliculogenesis is a complex and dynamic process by which follicles grow and develop with the goal of releasing a fertilizable oocyte upon ovulation. At birth, most females have a large pool of follicles; however, this pool is naturally depleted over time until ovarian senescence occurs and ovulation ceases. Thus, the population of preantral follicles is an appealing target for studies focused on female fertility, with the hopes of extending female reproductive lifespan. The studies within this thesis characterize, for the first time in mares, the population and spatial distribution of preantral follicles within the ovary and provide preliminary results documenting the success of a novel ovarian tissue transplantation site. The results from these studies showed that the population of preantral follicles in the mare ovary is similar to that of other species and that these follicles cluster in the ovary, comparable to mice and women. Furthermore, effects of age were observed, with young mares showing (i) a higher preantral follicle population, (ii) increased preantral follicle density in portions and regions, (iii) increased follicular clustering, specifically in the lateral and dorsal area of the ovary, and (iv) more numbers of neighbors per follicle than old mares. Additionally, interesting effects of spatial distribution in the ovary were also observed, with morphologically abnormal follicles in the intermediary portion being closest to the ovarian geometric center and with a tendency for increased follicular clustering in the ventral region. Furthermore, preantral follicles with neighbors were more likely to be morphologically normal. Moreover, despite having increased odds of lacking neighbors, it was observed that morphologically normal activated follicles had a higher number of neighbors than normal resting follicles. Lastly, the novel subvulvar mucosa was comparable to the established intramuscular location for heterotopic ovarian tissue transplantation, producing similar findings for macroscopic graft appearance, follicular density, and percentages of developing/growing follicles. The findings from these studies provide vital advancements in scientific understanding of preantral folliculogenesis in the mare and build important foundations for continuing to study folliculogenesis and treatments for infertility using the mare as an animal model.

O Brasil é o país que mais realiza aspiração folicular de oócitos guiada por ultrasonografia (OPU) para fins de produção in vitro de embriões bovinos. Com isso é possível aumentar consideravelmente o número de progênies por fêmea por ano. O objetivo desse trabalho foi identificar polimorfismos em genes envolvidos na foliculogênese e associar alterações gênicas com o número de folículos pré-antrais recrutados em bovinos, estimados pelo número de oócitos recuperados por OPU. Dados de aspiração folicular feitas a campo em fêmeas Nelore (Bos taurus indicus) foram obtidos de duas empresas diferentes, sendo que 30 fêmeas foram utilizadas para a prospecção de polimorfismos de nucleotídeos únicos (SNPs), 218 para estudos populacionais e dados de 193 fêmeas foram utilizados para a análise de efeitos das variações genéticas com o número de oócitos viáveis colhidos por OPU. Vinte fêmeas da raça Holandesa (Bos taurus taurus) foram genotipadas para a identificação de alelos taurinos ou zebuínos específicos. Regiões dos genes Gdf9, Fgf8, Fgf10 e BmprII foram amplificadas por meio da reação em cadeia da polimerase (PCR) e seqüenciadas para prospecção de SNPs. Quatro polimorfismos foram genotipados por meio de restrição enzimática de DNA, (PCR-RFLP; genes: Gdf9, Fgf8, Lhr e DNA mitocondrial-mtDNA) ou PCR em tempo real utilizando sondas específicas para cada alelo (BmprII). A avaliação de efeitos dos marcadores sobre a característica foi realizada por análise de variância utilizando modelos mistos com dados repetidos, em que o número de oócitos viáveis foi considerado variável dependente, as fêmeas foram consideradas variável aleatória, e local de aspiração, ano de aspiração e o SNP foram efeitos fixos. O mesmo modelo foi utilizado para estimar efeito médio de substituição alélica, em que o marcador foi substituído por uma co-variável numérica. A diferença entre médias de quadrados mínimos foram estimadas por meio de contrastes e a significância avaliada por teste t. Os efeitos ou as diferenças entre as médias foram consideradas significativas quando P<0,05. Foram identificados 19 SNPs sendo que 10 polimórficos entre as fêmeas Nelore, 04 causadores de substituição de aminoácidos na proteína. Entre os marcadores nucleares genotipados (Gdf9, A318C; Fgf8, C1027G; BmprII, A40048G; Lhr C62478T), todos apresentaram equilíbrio de segregação alélica dentro do marcador e genotípica entre os marcadores (P>0,05). Os SNPs nos genes Gdf9, BmprII e Lhr genotipados em fêmeas Nelore e Holandesas apresentaram-se fixados na segunda raça. Foram identificados efeitos dos polimorfismos dos genes Gdf9, Fgf8, BmprII e Lhr (P<0,05), entretanto os variantes de mtDNA não foram significativos. As diferenças entre médias confirmaram os resultados da ANOVA. Para os polimorfismos no gene Gdf9, BmprII e Lhr, observou-se a tendência de os indivíduos heterozigotos terem menores médias de oócitos viáveis quando comparados aos homozigotos, já o polimorfismo no gene Fgf8, observou-se a tendência de interação aditiva entre os alelos, em que foi estimado um efeito médio de 1,13±0,01 oócitos viáveis por troca de alelo C por G. Conclui-se que variações genéticas são causas de variabilidade do número de folículos presentes nos ovários, estimados por OPU. / Brazil is the country with the biggest number of ovum pick up (OPU) collection for bovine in vitro embryo production. This strategy is used to increase the number of calves born per cow per year. The objective of this work was to identify polymorphisms in genes related in folliculogenesis and associate some of the resulted gene variants with the number of pre antral follicle recruited in bovines, estimated by the number of oocytes collected by OPU. Records of open filed OPUs performed in Nelore cows (Bos taurus indicus) were obtained from two independent enterprises, and 30 females were used for single nucleotide polymorphism (SNP) prospection, 218 females for genotype and allelic population analysis and 193 females used for identification of genetic variation effects over the number of viable oocytes collected by OPU. Twenty Holstein cows (Bos taurus taurus) were also genotyped for identification of taurine and zebuine specific alleles. Segments of genes Gdf9, Fgf8, Fgf10 and BmprII were amplified by polymerase chain reaction (PCR) and sequenced for SNP prospection. Four polymorphisms were genotyped by means of enzymatic DNA digestion (PCR-RFLP, genes: Gdf9, Fgf8, Lhr and mitochondrial DNA - mtDNA) or real time PCR using allelic specific probes (Gene: BmprII). Genetic marker effect over the analyzed characteristic was realized by analysis of variance using mixed models with repeated data, in which the number of viable oocytes were set as dependent variable, the females were considered random variable, OPU place, OPU year and SNP were fixed effects. The same model was used for average allelic substitution effect estimation, where the genetic marker class was changed by a numeric covariant. The least square means (LSM) differences were estimated by contrasts and the significance evaluated by t test. Fixed effects or LSMs differences were considered significant when P<0.05. Nineteen SNPs were identified, from which ten were polymorphic among Nelore cows, four were expected to cause amino acid changes in protein. Among the nuclear genome markers genotyped (Gdf9, A318C; Fgf8, C1027G; BmprII, A40048G; Lhr C62478T, all resulted in allelic segregation equilibrium within marker and genotypic segregation equilibrium between markers (P>0.05). The SNPs in genes Gdf9, BmprII and Lhr genotyped in Nelore and Holstein cows were fixed in the last breed. SNPs in genes Gdf9, Fgf8, BmprII and Lhr affected the characteristic (P<0.05), however the mtDNA variants did not. The LSMs differences confirmed the ANOVA results. Heterozygote females for SNPs in genes Gdf9, BmprII and Lhr tend to have smaller LSMs than homozygous ones. On the other hand, there is indication of additive allelic interaction between alleles of SNP in gene Fgf8, for which an average allele substitution was estimated in 1.13±0.01 viable oocytes for a C/G change. We concluded that genetic variations were responsible for variable number of follicles present in ovaries, estimated by OPU.

Mammalian ovulation is a complex, hormone-dependent developmental program in which several events must take place in an ordered progression to ensure that the oocyte is competent for fertilization. This process requires the coordinated expression of many genes which must be turned on and off in the right place at the right time for proper development of the follicle. While the hormone signals from the brain that initiate ovulation are known, the master control genes which regulate this process are not well known. Homeobox proteins are potential candidates to perform as master regulators. Homeobox proteins are DNA-binding proteins that regulate the transcription of downstream genes and thereby control biological events. We recently identified a new homeobox gene cluster on the mouse X chromosome that are only expressed in reproductive tissues. These reproductive homeobox (Rhox) homeobox genes are expressed in the ovary, placenta, testis, and epididymis, and thus are good candidates to regulate both male and female reproductive tissue development and physiology. Rhox gene expression fell into three categories: Class I exhibited peak expression prior to ovulation (0-8 hours after hCG), Class II were predominantly expressed during ovulation (8-16 hours after hCG), and Class III peaked after ovulation. The slightly overlapping windows of peak Rhox gene expression suggest that these genes may regulate specific events during the ovulatory cycle. The founding member of the cluster, Rhox5, is highly expressed in granulosa cells of pre-ovulatory follicles. We previously reported that Rhox5-null female mice are viable and fertile, suggesting that RHOX5 is either not essential for ovulation, or that one of the other RHOX factors may compensate functionally in granulosa cells. In order to identify potentially redundant RHOX factors, we examined the expression patterns of all 32 Rhox genes using an eCG primed, hCG induced superovulation model, in wild-type, Rhox5-null, and heterozygous littermate mice. Expression levels of Rhox1, exhibited peak expression prior to being hormonal primed, was reduced in the Rhox5-null animal. However, Rhox8 mRNA and protein were reduced at 2h and 4h post hCG, but recovered once the follicles passed the antral stage of development. Conversely, in progesterone receptor knockout mice (PRKO), Rhox8 exhibited normal stimulation by eCG, but failed to reach its peak mRNA level at 8h post-hCG found in WT mice. This suggests a model in which Rhox8 transcription is dependent upon RHOX5 during early folliculogenesis and progesterone during the periovulatory window when RHOX5 normally wanes. Subsequent promoter analysis in granulosa cells revealed essential homeobox binding and progesterone response elements within Rhox8's 5'-flanking region. Transfection of RHOX5 and PGR expression plasmids stimulated, whereas dominant negative and mutant constructs inhibited, Rhox8 promoter activity. At present, the specific impact of misregulation of Rhox5 and Rhox8 during early folliculogenesis is not known. However, follicle counts from serially sectioned ovaries, extirpated from normal cycling animals, indicated that Rhox5-null mice possess ~50% fewer follicles than heterozygous littermates. Loss of RHOX5 in Sertoli cells results in male subfertility characterized by poor germ cell survival due in part to the misregulation of metabolism promoting genes. One of these genes, Ins2, is also stimulated by RHOX5 and RHOX8 in granulosa cells, suggesting impaired insulin signaling may contribute reduced follicle development in Rhox5-null ovaries.

The mammalian ovary contains a vast reserve of oocytes, most of which are enclosed within preantral follicles. A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. BPU fragments were collected from 5 to 21 yr old mares during the breeding season and processed using a tissue chopper, submitted to histological analysis or submitted to in vitro culture for 1 or 7 days. In conclusion, the BPU method is a reliable way to repeatedly harvest large numbers of viable, morphologically normal preantral follicles from living mares without affecting ovarian function or general reproductive health. Additionally, equine preantral follicles undergo development and growth when submitted to in vitro culture in α-MEM for 7 days, with some follicles maintaining morphological normality throughout. These technologies could be used in the future to enable the use of numerous oocytes present within the equine ovary to preserve genetic material or for large-scale embryo production.

Orientador: Irani Quagio-Grassiotto / Banca: Luís Fernando Fávero / Banca: Nelsy Fenerich Verani / Resumo: Nas fêmeas dos Teleostei a produção ilimitada de oócitos deve-se à constante proliferação das oogônias situadas no epitélio germinativo das lamelas ovígeras. Ao entrarem em meiose dão origem aos oócitos que são envoltos por uma camada de células foliculares, formando os folículos. No interior dos folículos, os oócitos passam por diferentes estágios de desenvolvimento (crescimento primário e secundário/vitelogênese, e maturação) ao final dos quais estão prontos para a desova. Os folículos pós-ovulatórios regridem e os óocitos que não tiveram sucesso na ovulação entram em atresia. A compreensão de como ocorre a foliculogênese nos Teleostei é recente e as descrições existentes referem-se aos grupos mais derivados. Por outro lado, os diferentes estágios de desenvolvimento dos oócitos, numa nova visão, vêm sendo utilizados na descrição de diferentes classes reprodutivas, ao longo do processo de maturação gonadal que ocorre a cada ano. Para testar a aplicabilidade desses novos conceitos aos Teleostei mais basais, analisou-se a foliculogênese e procedeu-se a caracterização celular das classes reprodutivas em Pimelodus maculatus, através de parâmetros histológicos, ultraestruturais e imunocitoquímico. Em P. maculatus a proliferação mitótica das oogônias, ocorre sempre acima da membrana basal, dá origem a conjuntos de células que são gradual e individualmente envoltos pelas células epiteliais, formando um tipo de cisto. As oogônias no interior dos cistos, ou oogônias secundárias entram em meiose ou dividem novamente 1, 2, 3 ou mais vezes por mitose e entram em meiose dando origem aos ovócitos. O conjunto de células, oócitos envoltos pelas células originárias do epitélio, forma uma espécie de sáculo, que se projeta para o estroma, mas permanece conectado ao próprio epitélio, compartilhando a mesma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The unlimited production of oocytes in Teleostei females is due to the constant proliferation of the oogonia located in the germinal epithelium of ovigerous lamellae. Upon entering meiosis, oogonia give origin to the oocytes, which are surrounded by a layer of follicular cells and thus form the follicles. Within the follicles, oocytes undergo different developmental stages (primary and secondary growth/vitellogenesis, and maturation) followed by spawning. Post-ovulatory follicles regress and the oocytes that failed to ovulate enter atresia. The understanding of how folliculogenesis occurs in Teleostei has been recently achieved, and the descriptions available to date refer to the most derived groups. On the other hand, oocyte developmental stages have been used, under this new view, to describe the different reproductive classes seen along the gonadal maturation process that takes place every year. In order to test the applicability of these new concepts to the most basal Teleostei, this study aimed at analyzing folliculogenesis, and characterizing the cells found in these reproductive classes in Pimelodus maculatus, by using histological, ultrastructural and immunocitochemical parameters. In P. Maculatus, oogonia mitotic proliferation always occurs above the basement membrane, giving rise to cell sets which are gradually and individually surrounded by epithelial cells forming a sort of cyst. The oogonia located within the cysts, or secondary oogonia, either enter meiosis or divide again 1, 2, 3 or more times through mitosis, giving origin to oocyte. The cell set, oocytes surrounded by the cells originated from the epithelium, forms a sac-like structure that projects into the stroma, but remains connected to the epithelium sharing the same basement membrane. The early meiotic cells, which are small, have a basophilic nucleus, and a clear and scarce cytoplasm... (Complete abstract click electronic access below) / Mestre

O Brasil é o país que mais realiza aspiração folicular de oócitos guiada por ultrasonografia (OPU) para fins de produção in vitro de embriões bovinos. Com isso é possível aumentar consideravelmente o número de progênies por fêmea por ano. O objetivo desse trabalho foi identificar polimorfismos em genes envolvidos na foliculogênese e associar alterações gênicas com o número de folículos pré-antrais recrutados em bovinos, estimados pelo número de oócitos recuperados por OPU. Dados de aspiração folicular feitas a campo em fêmeas Nelore (Bos taurus indicus) foram obtidos de duas empresas diferentes, sendo que 30 fêmeas foram utilizadas para a prospecção de polimorfismos de nucleotídeos únicos (SNPs), 218 para estudos populacionais e dados de 193 fêmeas foram utilizados para a análise de efeitos das variações genéticas com o número de oócitos viáveis colhidos por OPU. Vinte fêmeas da raça Holandesa (Bos taurus taurus) foram genotipadas para a identificação de alelos taurinos ou zebuínos específicos. Regiões dos genes Gdf9, Fgf8, Fgf10 e BmprII foram amplificadas por meio da reação em cadeia da polimerase (PCR) e seqüenciadas para prospecção de SNPs. Quatro polimorfismos foram genotipados por meio de restrição enzimática de DNA, (PCR-RFLP; genes: Gdf9, Fgf8, Lhr e DNA mitocondrial-mtDNA) ou PCR em tempo real utilizando sondas específicas para cada alelo (BmprII). A avaliação de efeitos dos marcadores sobre a característica foi realizada por análise de variância utilizando modelos mistos com dados repetidos, em que o número de oócitos viáveis foi considerado variável dependente, as fêmeas foram consideradas variável aleatória, e local de aspiração, ano de aspiração e o SNP foram efeitos fixos. O mesmo modelo foi utilizado para estimar efeito médio de substituição alélica, em que o marcador foi substituído por uma co-variável numérica. A diferença entre médias de quadrados mínimos foram estimadas por meio de contrastes e a significância avaliada por teste t. Os efeitos ou as diferenças entre as médias foram consideradas significativas quando P<0,05. Foram identificados 19 SNPs sendo que 10 polimórficos entre as fêmeas Nelore, 04 causadores de substituição de aminoácidos na proteína. Entre os marcadores nucleares genotipados (Gdf9, A318C; Fgf8, C1027G; BmprII, A40048G; Lhr C62478T), todos apresentaram equilíbrio de segregação alélica dentro do marcador e genotípica entre os marcadores (P>0,05). Os SNPs nos genes Gdf9, BmprII e Lhr genotipados em fêmeas Nelore e Holandesas apresentaram-se fixados na segunda raça. Foram identificados efeitos dos polimorfismos dos genes Gdf9, Fgf8, BmprII e Lhr (P<0,05), entretanto os variantes de mtDNA não foram significativos. As diferenças entre médias confirmaram os resultados da ANOVA. Para os polimorfismos no gene Gdf9, BmprII e Lhr, observou-se a tendência de os indivíduos heterozigotos terem menores médias de oócitos viáveis quando comparados aos homozigotos, já o polimorfismo no gene Fgf8, observou-se a tendência de interação aditiva entre os alelos, em que foi estimado um efeito médio de 1,13±0,01 oócitos viáveis por troca de alelo C por G. Conclui-se que variações genéticas são causas de variabilidade do número de folículos presentes nos ovários, estimados por OPU. / Brazil is the country with the biggest number of ovum pick up (OPU) collection for bovine in vitro embryo production. This strategy is used to increase the number of calves born per cow per year. The objective of this work was to identify polymorphisms in genes related in folliculogenesis and associate some of the resulted gene variants with the number of pre antral follicle recruited in bovines, estimated by the number of oocytes collected by OPU. Records of open filed OPUs performed in Nelore cows (Bos taurus indicus) were obtained from two independent enterprises, and 30 females were used for single nucleotide polymorphism (SNP) prospection, 218 females for genotype and allelic population analysis and 193 females used for identification of genetic variation effects over the number of viable oocytes collected by OPU. Twenty Holstein cows (Bos taurus taurus) were also genotyped for identification of taurine and zebuine specific alleles. Segments of genes Gdf9, Fgf8, Fgf10 and BmprII were amplified by polymerase chain reaction (PCR) and sequenced for SNP prospection. Four polymorphisms were genotyped by means of enzymatic DNA digestion (PCR-RFLP, genes: Gdf9, Fgf8, Lhr and mitochondrial DNA - mtDNA) or real time PCR using allelic specific probes (Gene: BmprII). Genetic marker effect over the analyzed characteristic was realized by analysis of variance using mixed models with repeated data, in which the number of viable oocytes were set as dependent variable, the females were considered random variable, OPU place, OPU year and SNP were fixed effects. The same model was used for average allelic substitution effect estimation, where the genetic marker class was changed by a numeric covariant. The least square means (LSM) differences were estimated by contrasts and the significance evaluated by t test. Fixed effects or LSMs differences were considered significant when P<0.05. Nineteen SNPs were identified, from which ten were polymorphic among Nelore cows, four were expected to cause amino acid changes in protein. Among the nuclear genome markers genotyped (Gdf9, A318C; Fgf8, C1027G; BmprII, A40048G; Lhr C62478T, all resulted in allelic segregation equilibrium within marker and genotypic segregation equilibrium between markers (P>0.05). The SNPs in genes Gdf9, BmprII and Lhr genotyped in Nelore and Holstein cows were fixed in the last breed. SNPs in genes Gdf9, Fgf8, BmprII and Lhr affected the characteristic (P<0.05), however the mtDNA variants did not. The LSMs differences confirmed the ANOVA results. Heterozygote females for SNPs in genes Gdf9, BmprII and Lhr tend to have smaller LSMs than homozygous ones. On the other hand, there is indication of additive allelic interaction between alleles of SNP in gene Fgf8, for which an average allele substitution was estimated in 1.13±0.01 viable oocytes for a C/G change. We concluded that genetic variations were responsible for variable number of follicles present in ovaries, estimated by OPU.