For this thesis our objectives were to establish an efficient method of ovarian synchronization and superstimulation in bison, and determine the effects of gonadotropin treatments on oocyte collection efficiency and quality in bison. In the first study we conducted two experiments to develop an efficient protocol for synchronization of follicular wave emergence during the anovulatory season. In Experiment 1, we compared the synchronizing effect of follicular ablation (n = 9) and treatment with 2 mg estradiol (E-) 17β in oil (n = 10), while in Experiment 2, we compared follicular ablation (n = 9) and treatment with 2 mg E-17β + 100 mg progesterone (P4; n = 10). Results showed that the degree of synchrony did not differ between ablation and hormone treatment groups in either Experiment, but follicular wave emergence was more synchronous in both treatment groups compared to the untreated control phase. The second study was conducted to develop an efficient method for ovarian superstimulation and oocyte collection during the anovulatory and ovulatory seasons. During the anovulatory season, one experiment was conducted in two replicates to compare the superstimulatory effect of 2500 IU of eCG (n = 10) given intramuscularly vs two doses of 200 mg of pFSH each (n = 10) given subcutaneously. Additionally, the effect of 25 mg of pLH given 24 hours prior oocyte collection on oocyte quality and collection rate was evaluated for each superstimulatory treatment. Results showed that treatment with pFSH induced a higher superstimulatory response and more cumulus oocyte complexes (COC) collected than did eCG during the anovulatory season. Furthermore, treatment with pLH increased the proportion of expanded COC that were collected with ultrasound-guided follicular aspiration. Two experiments were conducted during the ovulatory season, to develop an efficient protocol for superstimulation and oocyte collection. In Experiment 1, we compared the effect of two intramuscular doses of 200 mg of pFSH in saline (n = 11) vs two intramuscular doses of 200 mg of pFSH in a proprietary slow release formulation (SRF; n = 11). In Experiment 2, we compared the effect of a single dose of 2500 IU eCG intramuscularly vs two doses of 200 mg of pFSH administered subcutaneously. Results showed that a 2-dose regime of pFSH, diluted in either saline or a slow-release formulation induced a similar superstimulatory ovarian response in wood bison, while bison given a single-dose of 2500 IU eCG had a significantly lower ovarian response. In summary, synchronization of follicle wave emergence can be effectively accomplished in wood bison during the anovulatory season and follicular ablation, E-17β and E-17β + P4 treatments all shortened, and decreased the variability in the interval to follicular wave emergence. In addition, oocyte collection by transvaginal ultrasound-guided follicle aspiration from superstimulated bison was feasible and practical. Finally, treatment with pFSH was more effective than eCG to induce ovarian superstimulation for ultrasound-guided follicle aspiration in wood bison during both the anovulatory and ovulatory seasons.

The focus of the present thesis was on ultrasonographic, endocrine and molecular characterization of ovarian antral follicular waves in sheep. Transrectal ultrasonography and computer assisted image analysis were used to determine the feasibility of detecting ovulation and the forming corpus luteum (CL) and to non-invasively monitor CL differentiation and growth. High resolution transrectal ultrasonography and hormone measurements were used to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes and to correlate pulsatile secretion of gonadotropins with follicular growth in a wave, during the mid to late-luteal phase of the ovine estrous cycle. A series of experiments were conducted, using treatment with injections of ovine follicle stimulating hormone (oFSH) and measurement of serum concentrations of FSH, in cyclic and anestrous sheep, to investigate the existence of follicular dominance. We also evaluated the characteristics of secretory patterns of FSH that are critical for follicular wave emergence, in anestrous ewes. The possible existence of an endogenous rhythm of FSH secretion, independent of ovarian antral follicular dynamics, was studied in ovariectomized ewes. Finally, ovarian antral follicles at defined stages of growth in a follicular wave (based on transrectal ultrasonographic observations) were collected from separate groups of sheep by ovariectomy, to profile the expression patterns of steroidogenic enzymes (3¦Â-HSD, 17¦Á-OH and aromatase) using immunohistochemistry and gray-scale densitometric analysis. <p> The results of the present studies showed that it is possible to detect ovulation and visualize developing CL as early as 12-24 h after ovulation in the ewe. Changes in echotexture of the CL were closely associated with its morphological and functional characteristics, and we concluded that computer assisted image analysis holds promise for the noninvasive monitoring of CL differentiation and growth. Follicles reaching ovulatory diameter (¡Ý 5 mm) emerged and grew in a wave-like pattern in sheep, but without variation in the number of small follicles (1-3 mm in diameter), as seen in cattle. We concluded that all follicles that are recruited to grow beyond 2-3-mm in diameter, to 4-mm diameter in a wave, succeed in reaching an ovulatory diameter of ¡Ý 5 mm in the ewe. The emergence and growth of ovarian antral follicles in follicular waves, in sheep, do not require changes in LH secretion and may perhaps involve changes in the follicular sensitivity to LH. The largest follicle of a wave, in sheep, appears to have limited effects on other small follicles and on the time of emergence of the next follicular wave. Thus, functional dominance, as is present in cattle, may be absent in sheep. An endogenous rhythm for periodic peaks in serum FSH concentrations that is independent of ovarian follicular dynamics may exist in sheep. The expression patterns of steroidogenic enzymes, in the theca and granulosa compartments of antral follicles growing in each follicular wave in the ewe, paralleled serum estradiol concentrations, with the exception of the concentrations of 3¦Â-HSD in granulosa cells, which increased continuously from follicles 3 mm in diameter to the preovulatory follicle after the LH surge. The largest follicle of any follicular wave, irrespective of the stage of the cycle, would appear to be mature enough to ovulate if a gonadotropin surge is provided.

B cell development is a highly organized process, which commences in the fetal liver during embryogenesis and in the bone marrow (BM) after birth. Surface IgM+ immature B cells emigrate from the BM via the blood stream to the spleen and finally differentiate into conventional mature follicular B (FoB) cells and marginal zone (MZ) B cells. Conversely, some sIgM+ immature B cells can also mature into IgD+ FoB cells in the BM. The ubiquitously expressed cell surface glycoprotein CD47 and its receptor signal regulatory protein α (SIRPα) are members of the immunoglobulin superfamily. Both individually and upon their interaction, CD47 and SIRPα have been found to play important role in the homeostasis of T lymphocytes or CD8­ conventional dendritic cells (cDCs) in secondary lymphoid organs. However, their role in regulating B cell homeostasis has remained unknown. The present study describes important roles of CD47 and SIRPα in B cell homeostasis. Lack of SIRPα signaling in adult SIRPα mutant (MT - cytoplasmic domain deletion) mice resulted in an impaired B cell maturation in the BM and spleen, which was also reflected in the blood. In the BM and spleen of SIRPα MT mice, reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells were observed, while earlier BM B cell progenitors or splenic transitional B cells remained unaltered. In SIRPα MT mice, maturing B cells in BM and spleen were found to express higher levels of the pro-apoptotic protein BIM and contained an increased level of apoptotic cells. In contrast to that for FoB cells, the splenic MZ B cell population was increased with age in SIRPα MT mice without showing an increased level of activation markers. Immunohistochemical analysis revealed an increased follicular localization of MZ B cells in the spleens of SIRPα MT mice. In addition, MZ macrophages and marginal metallophilic macrophages were not restricted to their normal position in SIRPα MT spleens. Interestingly, CD47-deficient (CD47-/-) mice mimicked the FoB cell phenotype observed in SIRPα MT mice and had a reduced number of  FoB cells in the BM, blood and the spleen at 5­6 months of age, but not in younger mice. Similar to SIRPα MT mice, CD47-/- mice also displayed an increased number of splenic MZ B cells. Sera form both mouse strains did not show any signs of an increased production of autoantibodies or antinuclear antigens. BM reconstitution experiments identified a requirement for non-hematopoietic SIRPα signaling for normal B cell maturation in the BM and to maintain normal numbers and retention of MZ B cells in the splenic MZ. On the contrary, hematopoietic SIRPα signaling appeared to be important for FoB cell maturation in the spleen. Interestingly, hematopoietic SIRPα was required for normal MZ retention of MZ macrophages while normal distribution of metallophilic macrophages required non­hematopoietic SIRPα signaling.  Collectively, these findings revealed an important role of CD47 and of SIRPα signaling in B cell homeostasis in different lymphoid organs.

In recent decades salmonellosis has been on the rise as a food related illness worldwide. Causing over 24% of all non-typhoidal Salmonellosis cases, SE is the most frequently isolated serovar of Salmonella. Increased isolation of SE from eggs has paralleled an increase in the number of transovarian infections associated with laying hens in the poultry industry. This route of infection is a fairly new line of study when compared to the more traditional path where SE originates from fecal contamination through the shell. Salmonella Typhimurium (ST) is another concern for the egg industry. ST has caused 23.5% of all non-typhoidal salmonellosis cases. Understanding these two egg pathogens requires an in depth look at the mechanisms by which an egg may support infection and bacterial growth. Eggs were inoculated with both SE and ST onto the vitelline membrane and incubated for 24 hours. It was hoped that by gathering samples from the interior of the egg membrane, the albumen of the egg, and the membrane itself, some clarification as to when Salmonella is allowed to grow within the egg could be gathered. Albumen and membrane were found to be more hospitable environments to bacterial growth with increased storage times. In order to better understand the movement of bacteria into pre-ovulatory tissues, samples were gathered from mature laying hens. Follicular tissues were separated into divisions based on maturity, and bacteria were added to an in vitro cell culture broth containing the follicles. The point of this experiment was to determine if either species of Salmonella preferentially moved into follicles of different maturity when inoculated in vitro. A third experiment looked into the role of developmental stages of the vitelline membrane in exclusion of bacteria from the nutrient rich yolk. Tissues were gathered in the method described above. The follicular sack was removed from half of these samples and left intact for the other half. Another treatment group included was the yolks of eggs which had been laid by the same flock of birds. Results showed that follicles with intact follicular sacks were more susceptible to bacterial colonization than other treatment groups.

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.

Orientador: Cezinande de Meira / Banca: João Carlos Pinheiro Ferreira / Banca: Césa Ferraz Jacob / Resumo: O presente estudo teve como objetivo verificar o efeito da aspiração de folículos 25mm sobre a concentração de progesterona plasmática em éguas. O crescimento folicular foi acompanhado diariamente e um único folículo ovariano foi aspirado, exceto quando uma co-dominância foi observada. Neste caso, ambos os folículos foram aspirados no mesmo momento quando atingiram o diâmetro esperado nos respectivos grupos: F ³25mm (n = 6), F ³30mm (n = 6), F ³35mm (n = 6), F pré-ovulatório (n = 6). O Grupo controle (n = 5) não foi submetido à aspiração, acompanhando-se as ovulações espontâneas. A avaliação ultrassonográfica dos ovários foi realizada a cada 24 horas iniciando-se 48 horas antes da aspiração folicular (D0 = dia da aspiração), continuando até a ovulação subsequente. Para a análise estatística, foi utilizada a análise de variância de perfil seguida do método de Tukey com nível de significância de 5% para todas as variáveis, exceto: (1) animais que responderam à aspiração folicular atingindo concentrações de 2ng/mL de progesterona; (2) presença ou ausência de estrutura lútea visualizada pela ultrassonografia, onde foi utilizado o teste do qui-quadrado. A concentração de progesterona foi >2ng/mL, entre os dias 4,0±0,4 e 7,3±0,5 após a aspiração. O intervalo entre a aspiração e a luteólise foi de 16,0±0,5 a 19,0±1,4 dias e entre a aspiração e a ovulação foi de 17,2±2,8 a 23±0,5 dias. A concentração máxima de progesterona alcançada variou entre 6,4±2,6 e 10,9±1,8ng/mL e ocorreu entre os dias 8,7±3,4 e 11,5±1,2. A aspiração folicular em todos os grupos permitiu que as células foliculares se transformassem em estrutura hormonalmente ativa, produtora de progesterona em níveis compatíveis com o diestro. / Abstract: The present study aims to verify the follicles 25mm ablation effect on plasmatic progesterone concentration in mares. Follicular growth was daily accompanied and only one follicle was ablated, excepted when codominance was observed. At this case, both follicles were ablated at the same moment when achived the expected diameter for the following groups: F ³25mm (n = 6), F ³30mm (n = 6), F ³35mm (n = 6), F pre-ovulatory (n = 6). Control group (n = 5) was not submitted to ablation and the spontaneous ovulations were followed. Ultrasound evaluations of the ovaries were done every 24h beginning 48h after follicular ablations (D0 = day of ablation), continuing until subsequent ovulation. For statistical analysis, profile analysis followed by Tuckey method was used with a significance level of 5% for all variables excepted for: (1) animals that responded to follicular ablation achieving progesterone concentrations of 2ng/mL; (2) presence or absence of a luteum structure observed by ultrasound were analysed by Qui-square test. Interval between aspiration and luteolysis varied from 16,0±0,5 to 19,0±1,4 days and between ablation and next ovulation varied from 17,2±2,8 to 23±0,5 days. Maximum concentration of progesterone varied from 6,4±2,6 and 10,9±1,8ng/mL and occurred between days 8,7±3,4 and 11,5±1,2. In all groups, follicular ablation allowed follicular cells to become an active hormonal structure, which produced progesterone in concentrations similar to diestrous. / Mestre

Understanding the fundamental physiological mechanisms underlying ovarian physiology is important for accomplishing ways to diagnose and treat ovulatory dysfunction. With the advancement of assisted reproductive technologies, it became imperative to understand the details of the factors that affect the follicular development, oocyte competence, and embryo production. In primates and large domestic species including women and mares, ovarian follicles grow in a wave-like fashion with waves that can be anovulatory or ovulatory. Although considerable progress has been made in understanding ovarian follicular dynamics, more knowledge needs to be acquired to treat ovulatory problems like luteinized unruptured follicle (LUF) syndrome. Such information can be applied to optimizing treatment outcomes through assisted reproductive technologies that can be of benefit to both humans and animals. Three studies involving five experiments were conducted to improve our understanding on: 1) follicular growth patterns and endocrine profiles of LUFs and dominant follicles of major anovulatory and ovulatory waves in women; 2) physiologic mechanisms underlying luteinized unruptured follicles (LUFs) in mares with the goal of providing a model for human anovulation; and 3) dynamic changes in intrafollicular growth factors and hormones during the dominance and impending ovulation phases in mares. The results from Study 1 indicated that growth and endocrinological differences existed among LUFs, and physiological anovulatory and ovulatory dominant follicles in women. These differences were observed in follicle growth rates, intervals between follicular events, and in systemic concentration of hormones. In Study 2, LUFs were induced in 100% of the mares evaluated using a prostaglandin synthesis inhibitor; differences in intrafollicular prostaglandins, estradiol, growth hormone (GH), cortisol, and PRL were observed. In Study 3, changes in the concentrations of intrafollicular growth factors and hormones such as insulin-like growth factor-1 (IGF-1), inhibin-A, activin-A, VEGF, and PRL were detected during the dominance and impending ovulation phases in mares. The above-mentioned findings will provide the basis to compare the intrafollicular growth factors and hormones of potential LUFs and normal ovulatory follicles in the future. In conclusion, the results from these studies will shed light in the design of future studies on mechanisms of LUF formation and the role of intrafollicular growth factors and hormones in ovulation in women, mares, and perhaps also in other species.

Humoral immunity provides protection against subsequent infections. Antigen-specific, high-affinity, class-switched antibodies are produced by B cells through rounds of proliferation, B cell receptor rearrangement and selection in the germinal centres (GC). T cells play an essential and indispensable role in this process and in the recent years the term T follicular helper cells (TFH) was coined to describe this cell subset. The aim of my thesis is to investigate whether there is more than one type of T cells within the TFH population and whether it has important functional consequences. Firstly, I use sheep red blood cell immunisation (SRBC) and Salmonella enterica infection to show phenotypical differences between TFH expressing high and low level of surface molecule PD-1. In order to investigate the relationship between different TFH populations gene profiling was carried out on the microarray platform. Detailed transcriptome analysis revealed the discrete nature of isolated TFH cell subsets and provided an overview of their genetic landscape. Secondly, I have investigated the dependence of TFH subsets on cognate interactions with B cell in SRBC model by generating BM chimeras. I have demonstrated that generation of PD-1HI TFH, but not of PD-1LO TFH, depends on antigen presentation by B cells. Furthermore, I have shown that provision of wild-type but not MHC II knock-out B cells rescues PD-1HI formation in BM chimeras after SRBC immunisation. Finally, I have explored plasticity within TFH subsets and showed that none of the populations is in a terminally differentiated state, as they can convert into one another. Thirdly, experiments with S. enterica model revealed that the absence of PD- 1HI TFH is independent of the splenic architecture disruption present within the first week of the response. Surprisingly, co-immunisation studies showed that PD-1HI population is not only present but even enhanced in the group which received both SRBC and S. enterica when compared to single immunisations. The work presented in the thesis documents that there is a significant and previously unappreciated heterogeneity within TFH subset. This knowledge is important for designing optimal vaccine strategies and treating autoimmune diseases, as in both processes the antibody production plays a crucial role and its manipulation (either enhancing or blocking antibody production, respectively) can significantly improve clinical interventions.

The aim of this study was to define the ovarian follicular reserve of wild African elephants in terms of its type of small follicles (SF), its establishment and distribution throughout the ovaries, and the change in numbers of SF in the embryo and fetus as well as throughout prepubertal and adult life. The large elephant population in Zimbabwe provided the opportunity to collect ovaries from elephants culled for management reasons and hunted professionally. In total, gross morphological and histological studies were done on the gonadal ridges from 5 embryos (76–96 days post conception) and ovaries from 11 fetuses (4.8–22.2 months), 29 prepubertal females (2 months–10 years), 24 adult females (11–55 years) and 7 aged females (56–70 years). Specimens were fixed in 4% buffered formalin before a series of 25 ìm thick sections were cut and examined using stereological protocols to count SF numbers in each section and thereby calculate the follicle reserve of the whole ovary. Prior to counting SF numbers, their distribution throughout the ovary was studied and the repeatability of counts was validated. Numbers of SF were highest in mid-term fetuses, lower in fetuses during the second half of gestation, even lower in calves younger than 4½ years, whereas the numbers in calves aged 4½–9 years were significantly higher than those in younger calves, and similar to what they were in late-term fetuses. The numbers of SF were substantially and highly significantly lower in elephant 10–15 years in age compared to calves aged 4½9 years, suggesting a reduction around puberty. Thereafter the ovarian reserve fell steadily until depletion around the age of 70 years. During adult life the ovarian reserve was composed of early-primary (EP) and true-primary (TP) follicles. By 45 years of age only TP follicles remained although these enabled oestrous cyclical activity for many more years; of 7 sets of ovaries recovered from females aged 57–70 years, 6 showed evidence of cyclical activity or pregnancy within the preceding 6 years. The study shows that EP and TP form the follicular reserve from before birth until 45 years, with TP forming the reserve thereafter, which depletes in some old elephants and persists to maximum life span in others. / Thesis (PhD)--University of Pretoria, 2012. / Production Animal Studies / unrestricted

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.