Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO2 and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R-2 = 63%); percentage of oocytes that interacted with spermatozoa and BCF (R-2 = 73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R-2 = 64%) and velocity straight line (VSL; R-2 = 64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions. (c) 2005 Elsevier B.V. All rights reserved.

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation (3) administration of EPE twice daily in successively decreasing doses. In the first experiment. 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of greater than or equal to35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation. or two doses of a GnRH-a agonist upon detection of a follicle greater than or equal to35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles greater than or equal to35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF(2gamma) and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF(2alpha), but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles :35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose. improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates. (C) 2002 Elsevier B.V. All rights reserved.

Avaliação da maturação nuclear in vitro de oócitos de gatas domésticas (Felis catus) pré-púberes e púberes

O objetivo do trabalho foi avaliar a taxa de maturação nuclear in vitro de oócitos provenientes de gatas doméstica púbere e pré-púbere. Foram utilizadas 15 fêmeas felinas, 10 púberes e 5 pré-púberes; sendo os oócitos obtidos por aspiração quantificados e classificados. Os oócitos classificados como excelentes e regulares foram reunidos em grupos de 10, em meio de cultura, recobertos em óleo mineral em Placas de Petri siliconizadas e descartáveis. Após permanência em estufa, a 38°C e 5% de CO2 por 48 horas, os oócitos foram submetidos a duas lavagens com solução de hialuronidase a 0,4%, fixados em metanol/acido acético e corados com orceína acética. A avaliação da configuração cromossômica de oócitos maturados in vitro resultou em 44,68% das células em metáfase II no grupo das fêmeas púberes e 25,32% no grupo das doadoras pré-púberes, indicando que a puberdade influencia a capacidade dos oócitos se desenvolverem in vitro.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 x g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 mu m in diameter, with a dark ooplasm surrounded by three-or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70 degrees C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO(2) at 38 degrees C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.

Ovarian activity reversibility after the use of deslorelin acetate as a short-term contraceptive in domestic queens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural and Ultrastructural Aspects of Folliculogenesis in Didelphis albiventris, the South-American Opossum

The ovarian histology, the structural and the ultrastructural characteristics of the folliculogenesis in Didelphis albiventris were described in detail. Recent studies suggest that methatherian mammals have unusual reproductive cycle but there are few informations regarding the marsupials reproductive life. Despite of the opossum folliculogenesis pattern resembles methatherian and eutherian pattern in many aspects, the analysis shows some peculiar features of the oocyte structure and ultrastructure that make available new data on the reproductive biology of marsupials.

Influence of Superovulatory Protocols on In Vitro Production of Nellore (Bos indicus) Embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals.Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLC gamma/Ca(2+) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. on the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action.Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cellsthrough direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 213, in bovine follicles

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

The Mechanism of Oocyte Activation Influences the Cell Cycle-Related Genes Expression During Bovine Preimplantation Development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructural aspects of oogenesis and oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The ultrastructural characteristics of the organelles present in Serrasalmus spilopleura oogonia and oocytes undergoing primary growth were described in detail, considering its role in the nuclear and cytoplasmic metabolic processes that occur in these cell types. Even though these cells do not significantly differ from those similar to them that are found in other teleost groups, the analysis of their ultrastructure makes available new data on the reproductive biology of Characiformes. (C) 2001 Harcourt Publishers Ltd.

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups. (c) 2005 Published by Elsevier Ltd.

Sperm storage structures in the ovary of Helicolenus dactylopterus dactylopterus (Teleostei : Scorpaenidae): An ultrastructural study

The ultrastructure of ovarian sperm storage of Helicolenus dactylopterus dactylopterus is described, before and after the spawning period. The spermatozoa remain inside cryptal structures that are situated in the interlamellar gaps and are connected to the ovarian lumen by a duct. This complex forms a highly specialised structure. During the long storage period, crypts are richly vascularised. Their surrounding simple epithelia have intercellular junctions that may serve to protect the spermatozoa from the female immune system. At the moment during which insemination of mature oocytes occurs, the sperm may be expelled from cryptal structures by means of a spasmodic contraction. During the post spawning period, residual spermatozoa that remain in the crypts are eliminated by cryptal phagocytes. At the end of the process the crypts contain only an amorphous material.