Morphological characterization of the ovary and vitellogenesis dynamics in the tick Amblyomma cajennense (Acari: Ixodidae)

In this work we describe the internal morphology of the female reproductive system of the cayenne tick Amblyomma cajennense. This system is represented by a panoistic ovary, which lacks nurse cells in the germarium. This ovary consists of a single tube, in which a large number of oocytes develop asynchronously, thus accompanying the processes of yolk deposition in the oocytes. The oocytes were classified into stages that varied from I to V, according to: cytoplasm appearance, presence of the germ vesicle, presence of yolk granules, and presence of chorion. The study of vitellogenesis dynamics suggest that the yolk elements are deposited in the oocyte following a preferencial sequence, in which the lipids are the first to appear, followed by proteins an finally by the carbohydrates. In this way the yolk of A. cajennense ticks have these three elements that may be free in the cytoplasm or chemically bounded forming glycoprotein or lipoprotein complexes.     

Influence of pressmud on the development of the ovary,oogenesis and the neurosecretory cells of the earthworm Eudrilus eugeniae (Kinberg)

The histology of brain neurosecretory cells and the development of the ovary and oocytes of preclitellate, early clitellate and late clitellate stages of Eudrilus eugeniae reared in clay loam soil and pressmud have been studied. E. eugeniae reared in pressmud showed earlier differentiation of lobules in the ovary, and increasedoocyte numbers with larger cellular and nuclear volumes than those reared in clay loam soil. This indicates the nutritional superiority of pressmud over clay loam soil in supporting gametogenesis. In the preclitellate stage, the brain neurosecretory cell types (A and B) are small and appear to be laden with secretory substances. The neurosecretory cells become vacuolated and show increased cellular and nuclear volumes and nucleocytoplasmic indices during the reproductively active clitellate stages. Secretory activity of neurosecretory cells occurs earlier in worms reared in nutritively rich pressmud which has greater amounts of nitrogens, phosphorus and sugar, than in worms reared in clay loam soil.     

Ovarian Structure and Oogenesis of Catfish Pimelodella vittata (Lütken, 1874) (Siluriformes,Heptapteridae)

The morphology of the ovaries and oogenesis of Pimelodella vittata were studied using anatomical and histological techniques to provide information of its reproductive biology. Eighty adult females were captured trimonthly during the period November 2005 to October 2006. The ovaries are paired, saculiform organs, which are coated with tunica albuginea and contain ovigerous lamellae, where the oocytes develop before being released into the ovarian lumen and following the ovarian duct until reaching the genital papilla. Oogenesis was divided into stages based on the alterations to the nucleus, ooplasm and surrounding follicular layers. Oogonia form groups from the germinal epithelium have asynchronous development and differentiate into initial perinucleolar oocytes. The formation of the zona pellucida is initiated in the advanced perinucleolar oocytes reaching a thickness of 1.46 ± 0.58 μm in the vitellogenic oocytes. The follicular cells are squamous in perinucleolar oocytes, become cubical in the pre‐vitellogenic oocytes and prismatic in the vitellogenic oocytes with a height of 11.20 ± 4.74 μm. The histochemical reactions indicate that zona pellucida, cortical alveoli and yolk globules contain neutral glycoproteins and the follicular cells contain neutral glycoproteins in association with carboxylated and sulphated glycoconjugates. Statistical analyses showed significant differences in the diameter of the oocytes and follicular cells height as oocytes matured. This study represents the first data about the ovarian structure and oogenesis of this species.     

Vitellogenesis in the tick Amblyomma triste (Koch, 1844) (Acari: Ixodidae) Role for pedicel cells

This study presents new information on the vitellogenesis of the tick Amblyomma triste. In this species, the ovary consists of a layer of epithelial cells, which form the ovarian wall, oogonia and developing oocytes; and the pedicel, a cellular structure that synthesizes and provides yolk precursors for developing oocytes. The pedicel also attaches oocytes to the external surface of the epithelial wall. In this study, evidence is provided in support of pedicel cells in providing compounds for oocytes during vitellogenesis.     

Light microscopical and ultrastructural characterization of black howler monkey (Alouatta caraya) ovarian follicles

The present study describes the morphological characteristics of black howler monkey (Alouatta caraya) ovarian follicles. One ovary of an adult healthy black howler monkey was collected and processed for light and electron microscopy. Primordial, primary, secondary, tertiary and pre-ovulatory follicles were evaluated for their morphometrical aspects. The ovary of black howler monkey presented a distinct conformation with a uniform distribution of the follicles mostly in the peripheric cortex. This black howler monkey ovary presented a total of 59 921 ovarian follicles. From this amount, 71.1% were classified as primordial, 18.9% as primary, 8.1% as secondary, 1.4% as tertiary and 0.5% as pre-ovulatory follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells are described. Moreover, primordial, primary and secondary follicles have been observed by electron microscopy.     

The surface features of the epithelial lining of the ducts of the epididymis of the ostrich (Struthio camelus)

The luminal appearance of the various ducts of the epididymis of the ostrich was studied by scanning electron microscopy in tissues fixed by immersion in glutaraldehyde. The ductal types were similar to those previously described for some other species of birds. Numerous short microvilli, as well as a single cilium, projected from the apical surface of the rete testis cell. The ciliated cells of the efferent ductules projected tufts of cilia into the ductal lumen, while the non-ciliated cells bore short microvilli. The connecting and epididymal ducts were lined by a columnar cell type whose apical surface bore uniformly distributed microvilli and a single, centrally situated cilium. The spermatozoa found in all ducts of the epididymis bore a distal cytoplasmic droplet. This observation has implications for the maturational process in the ostrich spermatozoon in the epididymis. The surface features of the ducts, except for a few noteworthy differences, were generally similar to those previously described for the male domestic fowl, turkey and duck.     

Oocyte ultrastructural characteristics in camel (Camelus dromedarius) primordial to large antral follicles

This study was conducted to describe in detail the ultrastructural features and morphological characteristics of camel oocytes from preantral follicles in relation to the sequential stages of follicular development and also for oocytes from antral follicles in relation to their diameter. Camel oocytes from primordial, primary, secondary and also early to late antral follicles were processed and examined by light and transmission electron microscopy. Primordial follicular oocytes were characterized by a layer of flattened granulosa cells around and also eccentric nucleus and few cytoplasmic organelles in the peripheral region. Up to the secondary follicle stage, flat cells were replaced by cuboidal granulosa cells and their number increased and also an increase in the number of organelles such as vesicles, mitochondria and endoplasmic reticulum was observed. In the early antral stage, the formation of zona pellucida, appearance of microvilli and pleomorphic mitochondria was seen and the nucleus was dislocated to the peripheral region. During final growth phase, the extent of endoplasmic reticulum, vesicles and mitochondria increased, the number of lipid droplets decreased and cumulus cell process endings (CCPE) were observed. In conclusion, the growth of camel oocyte is associated with progressive increase in the number of mitochondria, endoplasmic reticulum, Golgi complexes and cytoplasmic vesicles as well as decrease in the number of lipid droplets and the nucleus migration from an eccentric in preantral to a peripheral location in antral follicles.     

Follicle formation in the canine ovary after autografting to a peripheral site

This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.     

Interactions between the oocyte and surrounding somatic cells in follicular development: lessons from in vitro culture

Mammalian oogenesis occurs concomitantly with folliculogenesis in a coordinated manner in the ovaries. In vitro growth (IVG) culture systems of the oocytes have been developed as a new technology for utilizing incompetent oocytes in the ovary as a source of mature oocytes as well as for studying oogenesis, folliculogenesis, and oocyte-somatic cell interactions. The results of IVG experiments have suggested that direct association of oocytes and surrounding granulosa cells supports oocyte viability and growth through the gap junctions, which are efficient conduits for low molecular weight substances. It has been revealed that granulosa cells metabolize some molecules which are in turn transported into the oocytes. IVG systems have also provided evidence that FSH promotes the development of follicles at secondary or later stages by its stimulation of proliferation and differentiation of granulosa cells, and perhaps by its anti-apoptotic effects. In addition, interactions between granulosa cell-derived KIT ligands and oocyte KIT receptors have been suggested as initiating oocyte growth and follicular development. Furthermore, recent findings suggest there are growth factors derived from oocytes such as GDF-9 and BMP-15. With such factors, oocytes participate in follicular development by regulating the differentiation of surrounding somatic cells. These bidirectional communications between oocytes and somatic cells are important for oocyte growth and follicular development. IVG systems should provide further information regarding oogenesis and folliculogenesis in the ovary.     

Localization of thrombospondin‐1 and its receptor CD36 in the ovary of the ostrich (Struthio camelus)

Angiogenesis, the formation of new blood vessels from pre‐existing vasculature, plays a decisive role for the rapid growth of avian follicles. Compared to mammals, few data on the angiogenesis in the avian ovary are available. However, whereas several pro‐angiogenic factors in the avian ovary have been recently studied in detail, little information is available on the localization of anti‐angiogenic factors. The aim of this study was to determine the localization and possible function of the anti‐angiogenic factor thrombospondin‐1 (TSP‐1) and its receptor CD36 in the ovary of the ostrich using immunohistochemistry and to correlate the results with ultrastructural data. Whereas the oocytes and granulosa cells of all follicular stages were negative for TSP‐1, myofibroblasts of the theca externa and smooth muscle cells of blood vessels showed distinct reactions. A distinctly different staining pattern was observed for CD36. The oocytes were CD36 negative. No immunostaining for CD36 could be observed neither in the granulosa cells nor in the adjacent theca interna of vitellogenic follicles. In the theca externa, blood vessels protruding towards the oocyte showed CD36‐positive endothelial cells. In conclusion, a fine balance between angiogenic and anti‐angiogenic processes assures that a dense net of blood vessels develops during the rapid growth of a selected follicle. Anti‐angiogenic molecules, such as TSP‐1 and its receptor CD36 may, after the oocyte has reached its final size, inhibit further angiogenesis and limit the transport of yolk material to the mature oocyte. By this mechanism, the growth of the megalecithal oocyte during folliculogenesis may cease.     

Promotion of ovarian follicular development by injecting vascular endothelial growth factor (VEGF) and growth differentiation factor 9 (GDF-9) genes

Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles, indicating that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary. On the other hand, the development of antral follicles is associated with increased density of blood vessels within the theca cell layers surrounding the follicles. A recent study reported that vascular endothelial growth factor (VEGF) play an important role in the process of thecal angiogenesis during follicular development. To investigate whether additional induction of thecal angiogenesis would support subsequent follicular development, miniature gilts were directly injected VEGF gene into the ovary. Injection of VEGF gene increased the levels of mRNA expression of VEGF 120 and VEGF 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene compared with those treated with eCG alone, indicating that the regulation of thecal angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. This technique may be an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.     

Effects of the Reproductive Status on Morphological Oocyte Quality and Developmental Competence of Oocytes after In Vitro Fertilization and Somatic Cell Nuclear Transfer in Cat

This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p < 0.05). The proportions of IVF embryos cleaved and developed to blastocysts significantly decreased with decreased quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos.     

The Expression of FSH Receptor (FSHR) in the Neonatal Porcine Ovary and its Regulation by Flutamide

This study was designed to reveal the FSHR mRNA and protein expression in the neonatal porcine ovary and to determine whether maternal administration of antiandrogen flutamide may affect FSHR expression in the ovary of newborn piglets using real‐time PCR, immunohistochemistry and Western blot analysis. Pregnant sows were injected with flutamide at a dose of 50 mg/kg body weight, given five times, every second day, starting at day 20 post‐coitum (p.c.) or day 80 p.c., and ovaries were obtained from neonatal pigs. The FSHR mRNA expression was significantly decreased after flutamide administration. Furthermore, higher down‐regulation was observed following exposure to antiandrogen at day 20 than at day 80 p.c. Immunohistochemistry showed the positive immunostaining for FSHR in the oocytes, granulosa cells of primary follicles and the surface epithelium of the ovaries from both control and flutamide‐treated pigs. However, oocytes and granulosa cells of primary follicles in the ovaries exposed in utero to flutamide were weakly immunostained when compared to those in the control ones. The presence of FSHR protein in all investigated ovaries was confirmed by Western blot analysis. Based on our findings, we suggest that FSHR may be involved in the early follicle formation in pigs, which begins during prenatal life. Furthermore, the regulation of FSHR mRNA and protein expression in neonatal porcine ovaries after maternal exposure to flutamide confirms that androgens play a crucial role in porcine folliculogenesis at the early stages.     

Impact of compound alpha treatment in vivo on egg production by the liver fluke, Fasciola hepatica

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48 h and 72 h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis' gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs. Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48 h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72 h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48 h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72 h pt. In the Mehlis' gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48 h to 72 h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action.     

Immunolocalization and expression pattern of gpr3 in the ovary and its effect on proliferation of ovarian granulosa cells in pigs

Gpr3, a member of the G protein-coupled receptor superfamily, was known as a critical factor for the maintenance of meiotic prophase arrest in oocytes via a Gs protein-mediated pathway. The present studies were conducted to examine the ovarian immunolocalization of Gpr3, its expression pattern in different stages of fetal, postnatal and developmental pigs and its effect on proliferation of ovarian granulosa cells in pigs. Immunohistochemical analysis indicated that Gpr3 was localized in egg nests, oocytes and granulosa cells (GCs) of the follicle ranging from the primordial to Graafian stages and the corpora lutea. Staining was faintly present in the corpora lutea and weak in GCs but was strong in oocytes. Real-time PCR and Western blotting indicated that Gpr3 mRNA and protein were both present in the different ages of ovaries, and there were wavy changes in the expression levels from postpartum 1 to 180 days. Moreover, both the mRNA and protein levels of Gpr3 were upregulated significantly during follicle growth, suggesting that Gpr3 might play potential roles in regulating ovarian follicle development in the pig. MTT and flow cytometry analyses indicated that Gpr3 knockdown significantly promoted proliferation of porcine GCs while increasing the proportion of cells in the S phase and the expression of Cyclin B1 and Cyclin D2, providing new insights into how Gpr3 signaling regulates the proliferation of porcine GCs. In conclusion, the stage- and cell-specific expression pattern of Gpr3 in the porcine ovary suggested that Gpr3 might play an important role during the entire process of follicular development and luteinization.     

Microscopic Evidence of the Opportunistic Reproductive Strategy and Early Sexual Maturation of the Small‐Sized Characin Knodus moenkhausii (Characidae,Pisces)

We investigated the reproductive biology of Knodus moenkhausii, an abundant small‐sized characin fish with broad occurrence in the Paraná River basin, Brazil. Specimens were collected monthly to determine fecundity, length at first maturity, reproductive period and spawning type. Gonads were macroscopically classified according to their form, size and texture in three different stages (immature, maturing or mature). Histological procedures were conducted to confirm gonadal developmental stages, and it was possible to notice that maturing females actually presented atretic oocytes, and all males that were macroscopically classified as immature, maturing and mature actually presented abundant spermatozoa in their gonads. Because of these discrepancies, a reclassification of gonadal maturations stages was needed after histological analysis, reinforcing its importance to studies on the reproduction of small characins. Reproduction occurred throughout the year though with two peaks. The length of the smallest mature individuals was 13 mm SL for males and 24 mm SL for females. Despite presenting relatively small batch fecundity, some life history traits such as early reproduction, multiple spawning throughout the year, in association with known opportunistic feeding habits, explain the high abundance of this species in locations where it occurs.     

Conditions affecting growth and developmental competence of mammalian oocytes in vitro

Mammalian ovaries contain a large number of oocytes at different stages of growth. To utilize potential female gametes, it is important to develop culture systems that permit oocytes to achieve full growth and competence in order to undergo maturation, fertilization and development. The desired culture systems should meet at least the following three conditions: (i) oocytes remain healthy and functional so that they can execute intrinsic programs that direct their growth and development; (ii) granulosa cells that are adjacent to oocytes proliferate efficiently to prevent oocytes from becoming denuded; and (iii) granulosa cells maintain (and develop) appropriate associations with oocytes during the culture period. For this reason, several systems have been developed, and they can be classified into four categories based on the structure and components of the follicle/oocyte–granulosa cell complex and the location of the oocyte in the physical organization of the complex. The resultant diverse morphologies are due to multiple factors, including the method for initial isolation of follicles, the culture substrate, and hormones and other factors added into the medium. It is important to find an optimal combination of such factors involved in the process to facilitate future research efforts.     

Effect of Initial Cumulus Morphology on Meiotic Dynamic and Status of Mitochondria in Horse Oocytes during IVM

The aim of this investigation was to examine the chromatin configuration of the nucleus, pattern of mitochondrial aggregation and mitochondrial activity in parallel studies in the same horse oocytes. Horse oocytes recovered by ultrasound-guided follicle aspiration in vivo were classified according to two main initial cumulus morphologies as having compact or expanded cumulus. The percentage of oocytes with a diplotene meiotic configuration at the time of recovery from the follicles was highest in compact oocytes. Oocytes with expanded cumulus layers at the time of recovery matured more rapidly in vitro and reached a proportion >50% at the metaphase II stage (M 2) sooner during in vitro maturation (IVM), than did compact oocytes. The mitochondrial aggregation pattern changed from finely distributed (Type 1) through crystalline (Type 2) to an aggregated, granulated appearance (Type 3) during IVM. The pattern of mitochondrial aggregation at the time of recovery was associated with the initial cumulus morphology of the oocyte, in that compact oocytes had a higher proportion of Type 1 aggregation, whereas expanded oocytes had a higher proportion of Type 3. The fluorescence intensity of metabolic active mitochondria, measured by fluorescence intensity (Em 570) per oocyte after MitoTracker CMTM Ros orange labelling, increased in the oocytes during IVM and depended on initial cumulus investment. Oocytes with the granulated type of aggregated mitochondria Type 3 had the highest level of metabolic activity and were in more progressed stages of meiosis (A 1-M 2). Oocytes initially having expanded layers of cumulus reached significantly higher levels of mitochondrial activity after IVM than did oocytes initially having compact cumuli. During resumption of meiosis the mitochondrial activity of oocytes with initially expanded cumulus increased continuously up to M 2, whereas in oocytes from compact cumulus-oocyte complex (COC), the activity declined after A 1/T 1 stages of meiosis.