Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (> 18 mm) follicles. RNeasy Micro Kit (Qiagen(A (R))) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

Ultrastructure of oocytes of the Urostreptus atrobrunneus (Diplopoda, Spirostreptida, Spirostreptidae): A potential urban centers plague

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

Análise do período de defeso do camarão marinho Artemesia longinaris através da maturidade ovariana anual

Pós-graduação em Aquicultura - FCAV

Avaliação da atividade gonadal de machos e fêmeas de pescada branca, Plagioscion squamosissimus (Heckel, 1840), (Pisces, Teleostei, Sciaenidae) no rio Pará (PA)

Fêmeas e machos adultos de P. squamosissimus (Pisces, Teleostei, Sciaenidae) foram coletados mensalmente no Rio Pará, que banha a ilha do Capim (PA) (S 010 34. 971`; W 0480 52.932`), durante o período de fevereiro de 2004 a fevereiro de 2005, correspondendo ao total de 234 espécimes. As gônadas foram coletadas, fixadas e processadas de acordo com os métodos usuais utilizados para processamento em parafina e análise em microscopia de luz. A espécie em estudo é uma das principais fontes de proteína animal para a população local, sendo capturada de forma intensa e ininterrupta ao longo do ano. Com base em informações dos pescadores locais, essa captura parece estar determinando uma aparente diminuição quantitativa e qualitativa em seus estoques locais. A espécie apresenta desova parcelada e o desenvolvimento gonadal foi caracterizado nos estádios de repouso, maturação, maduro e esvaziado ou semi-esvaziado. Gônadas maduras foram encontradas nos meses de dezembro, janeiro e julho. A análise de correlação entre o estádio gonadal maduro e a variação temporal da relação gonadossomática (ΔRGS) indica também a ocorrência de desova nos respectivos meses. Não obstante, as informações obtidas da correlação entre a ΔRGS e a média de oócitos maduros e percentual de espermatozóides por túbulo seminífero, respectivamente, também indicaram haver desova nos respectivos meses, sendo que aparentemente a espécie apresenta uma desova mais intensa ou desova principal entre os meses de dezembro e janeiro (inverno), e outra desova menos intensa ou secundária no mês de julho (verão). Com base no método da morfologia tubular, foram determinados oito estádios do ciclo do epitélio seminífero (CES), sendo que no estádio 1 os túbulos seminíferos são compostos por espermatogônias primárias e cistos de spermatogônias secundárias; o estádio 2 é composto por espermatogônias primárias e secundárias e cistos de espermatócitos; estádio 3 é caracterizado por espermatogônias primárias, secundárias, cistos de espermatócitos e de espermátides jovens ou recém-formadas; estádio 4 com túbulos seminíferos caracterizados pela presença de espermatogônias primárias e secundárias, espermatócitos e por cistos de espermátides jovens e tardias; estádio 5 apresenta todas as células anteriores e é marcado pelo surgimento de espermatozóides no lúmen tubular; estádio 6 tem como características a diminuição dos cistos de células germinativas e considerável aumento do número de espermatozóides no lúmen tubular; os túbulos seminíferos no estádio 7 contêm poucos cistos de células germinativas e se inicia o esvaziamento da massa de espermatozóides do lúmen tubular; estádio 8 é o último do CES e é caracterizado pela aparente desorganização dos cistos remanescentes de células germinativas no túbulo seminífero.

Molecular basis of maturation promoting factor

Maturation promoting factor (MPF), which is functionally defined by its ability to induce Xenopus oocyte maturation, is an M phase (meiosis and mitosis) specific activity that is present in all species tested. It was hypothesized that MPF is a universal trigger of the interphase to M phase transition during the cell cycle. The current model for the molecular basis of MPF is that MPF is a protein kinase having the cdc2 protein as its catalytic subunit and is identical to the M phase-specific histone H1 kinase. In the present study, I have shown that more than just cdc2 kinase contributes to MPF activity, and M phase-specific H1 kinase is composed of at least two entities, instead of just cdc2 kinase. Therefore, the simple model of MPF = cdc2 kinase = M phase-specific H1 kinase should be ruled out.^ My study began with the characterization of the mitosis-specific monoclonal antibody MPM-2. MPM-2 reacts specifically with M phase cells from different species by recognizing a discrete set of proteins once they are phosphorylated at the G$\sb2$/M transition. I found that phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity during oocyte maturation stimulated by progesterone. If MPM-2 was injected into oocytes before the stimulation, MPF activity failed to appear, and the oocytes could not mature. Furthermore, MPM-2 was able to deplete MPF activity from M phase extracts. These results identified MPM-2 as a probe that recognizes either MPF itself or a regulator of MPF.^ Since M phase-specific H1 kinase was believed to be identical to cdc2 kinase and MPF, I proceeded to determine whether MPM-2 recognized the M phase-specific H1 kinase. I found that MPM-2 did recognize an M phase-specific H1 kinase. However, this kinase was not cdc2 kinase. This kinase (MPM-2 kinase) is present in a latent form in immature oocytes and is activated in tandem with the activation of MPF during oocyte maturation. It appears to accelerate progesterone-induced oocyte maturation. Therefore, MPM-2 kinase may be a novel positive regulator of MPF activation.^ MPM-2 depletes MPF activity, but not cdc2 kinase activity. This discrepancy caused me to question the equivalency of MPF with cdc2 kinase. I found that when a high percentage of MPF activity was recovered from gel filtration of mature oocyte extract, the recovered MPF activity was due to two factors, cdc2 kinase and a factor recognized by MPM-2. This factor might activate and stabilize cdc2 kinase. Identification of this factor in the present study may contribute to the understanding of the autoactivation of MPF. ^

Fire–diffuse–fire model of dynamics of intracellular calcium waves

When Ca2+ is released from internal stores in living cells, the resulting wave of increased concentration can travel without deformation (continuous propagation) or with burst-like behavior (saltatory propagation). We analyze the “fire–diffuse–fire” model in order to illuminate the differences between these two modes of propagation. We show that the Ca2+ release wave in immature Xenopus oocytes and cardiac myocytes is saltatory, whereas the fertilization wave in the mature oocyte is continuous.

Escala y talla de primera madurez gonadal en “NAVAJA” Tagelus dombeii (Lamarck, 1818), entre las zonas Parachique - Las Delicias, 2009

Para el estudio de la gametogénesis, escala de madurez y estimación de la talla de primera madurez gonadal de “navaja” Tagelus dombeii, las muestras procedieron de las zonas Caleta de Parachique y Las Delicias, y fueron colectadas a bordo de embarcaciones marisqueras por buzos artesanales, durante Enero – Diciembre del año 2009. La zona para tomar las muestras comprendieron desde los grados 05º 40' 33.3'' (LS) hasta 05º 49' 21.8'' (LS). En el estudio de la gametogénesis, se observó que los ovarios presentaron tres tipos de ovocitos: ovocito inmaduro (OI), ovocito en maduración (OEM) y ovocito maduro (OM), además del ovocito atrésico (OA) para las hembras, mientras que para los machos las células sexuales encontradas fueron: espermatogonio = SG, espermatocito = SC y espermatozoide = SP. La escala microscópica de madurez gonadal para “navaja” T. dombeii fue establecida con seis estadios, siendo estos: Virginal = 0, Reposo = I, En Maduración = II, Maduro =III, Desovante/Expulsante = IV y Recuperación = V. Así mismo, se estableció, que la talla de primera madurez gonadal es de 61 mm de longitud total (LT) para las hembras y 58 mm para machos, obteniendo un promedio de 58 mm LT. Mientras que la talla de primer desove/expulsión fue de: 67 mm (LT) para hembras y 66 mm LT para machos siendo promedio para ambos sexos de 66 mm LT. Además, se reporta la capacidad de T. dombeii de cambiar el sexo de hembra a macho (hermafroditismo protógino).

Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification

Objective: To compare cryopreservation of mature human oocytes with slow-rate freezing and vitrification and determine which is most efficient at establishing a pregnancy. Design: Prospective randomized. Setting: Academically affiliated, private fertility center. Patient(s): Consenting patients with concerns about embryo cryopreservation and more than nine mature oocytes at retrieval were randomized to slow-rate freezing or vitrification of supernumerary (more than nine) oocytes. Intervention(s): Oocytes were frozen or vitrified, and upon request oocytes were thawed or warmed, respectively. Main Outcome Measure(s): Oocyte survival, fertilization, embryo development, and clinical pregnancy. Result(s): Patient use has resulted in 30 thaws and 48 warmings. Women`s age at time of cryopreservation was similar. Oocyte survival was significantly higher following vitrification/warming (81%) compared with freezing/thawing (67%). Fertilization was more successful in oocytes vitrified/warmed compared with frozen/thawed. Fertilized oocytes from vitrification/warming had significantly better cleavage rates (84%) compared with freezing/thawing (71%) and resulted in embryos with significantly better morphology. Although similar numbers of embryos were transferred, embryos resulting from vitrified oocytes had significantly enhanced clinical (38%) pregnancy rates compared with embryos resulting from frozen oocyte (13%). Miscarriage and/or spontaneous abortion rates were similar. Conclusion(s): Our results suggest that vitrification/warming is currently the most efficient means of oocyte cryopreservation in relation to subsequent success in establishing pregnancy. (Fertil Steril (R) 2010; 94: 2088-95. (C) 2010 by American Society for Reproductive Medicine.)

Oocyte diameter as a predictor of fertilization and embryo quality in assisted reproduction cycles

Objective: To assess the impact of the mean oocyte diameter (MOD) on occurrence of fertilization and embryo quality in assisted reproduction cycles. Design: Prospective observational study. Setting: Sector of Human Reproduction of the University Hospital, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo (HCFMRP-USP). Patient(s): Thirty-five women undergoing intracytoplasmic sperm injection (ICSI) at the University Hospital of Ribeirao Preto from May to October 2007. Intervention(s): MOD assessment. Main Outcome Measure(s): Occurrence of fertilization and qualitative embryo classification on 2nd and 3rd day after ICSI. Result(s): We divided 160 metaphase II oocytes according to MOD into groups A (MOD below the 25th percentile), B (MOD between 25th and 75th percentile), and C (MOD above the 75th percentile). There was no statistically significant association between MOD and the occurrence of fertilization or the qualitative embryo classification on days 2 and 3. There was no statistically significant difference between groups regarding number of cells or the qualitative embryo classification on days 2 and 3. Conclusion(s): The MOD of mature oocytes does not seem to be related to the occurrence of fertilization or to the developmental quality of human embryos on days 2 and 3 after ICSI. (Fertil Steril(R) 2010;93:621-5. (C)2010 by American Society for Reproductive Medicine.)

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 mu g/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX-blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Estradiol and testosterone concentrations in follicular fluid as criteria to discriminate between mature and immature oocytes

The objective of the present study was to examine the association between follicular fluid (FF) steroid concentration and oocyte maturity and fertilization rates. Seventeen infertile patients were submitted to ovulation induction with urinary human follicle-stimulating hormone, human menopausal gonadotropin and human chorionic gonadotropin (hCG). A total of 107 follicles were aspirated after hCG administration, the oocytes were analyzed for maturity and 81 of them were incubated and inseminated in vitro. Progesterone, estradiol (E2), estrone, androstenedione, and testosterone were measured in the FF. E2 and testosterone levels were significantly higher in FF containing immature oocytes (median = 618.2 and 16 ng/ml, respectively) than in FF containing mature oocytes (median = 368 and 5.7 ng/ml, respectively; P < 0.05). Progesterone, androstenedione and estrone levels were not significantly different between mature and immature oocytes. The application of the receiver-operating characteristic curve statistical approach to determine the best cut-off point for the discrimination between mature and immature oocytes indicated levels of 505.8 ng/ml for E2 (81.0% sensitivity and 81.8% specificity) and of 10.4 ng/ml for testosterone (90.9% sensitivity and 82.4% specificity). Follicular diameter was associated negatively with E2 and testosterone levels in FF. There was a significant increase in progesterone/testosterone, progesterone/E2 and E2/testosterone ratios in FF containing mature oocytes, suggesting a reduction in conversion of C21 to C19, but not in aromatase activity. The overall fertility rate was 61% but there was no correlation between the steroid levels or their ratios and the fertilization rates. E2 and testosterone levels in FF may be used as a predictive parameter of oocyte maturity, but not for the in vitro fertilization rate.

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae)

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

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae)

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I-Ill and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two Or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption. (C) 2011 Elsevier GmbH. All rights reserved.

Xenotransplantation of cryopreserved human ovarian tissue--a systematic review of MII oocyte maturation and discussion of it as a realistic option for restoring fertility after cancer treatment.

OBJECTIVE To systematically review the reporting of MII (MII) oocyte development after xenotransplantation of human ovarian tissue. DESIGN Systematic review in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). SETTING Not applicable. PATIENT(S) Not applicable. INTERVENTION(S) Formation of MII oocytes after xenotransplantation of human ovarian tissue. MAIN OUTCOME MEASURE(S) Any outcome reported in Pubmed. RESULT(S) Six publications were identified that report on formation of MII oocytes after xenotransplantation of human ovarian tissue. CONCLUSION(S) Xenografting of human ovarian tissue has proved to be a useful model for examining ovarian function and follicle development in vivo. With human follicles that have matured through xenografting, the possibility of cancer transmission and relapse can also be eliminated, because cancer cells are not able to penetrate the zona pellucida. The reported studies have demonstrated that xenografted ovarian tissue from a range of species, including humans, can produce antral follicles that contain mature (MII) oocytes, and it has been shown that mice oocytes have the potential to give rise to live young. Although some ethical questions remain unresolved, xenotransplantation may be a promising method for restoring fertility. This review furthermore describes the value of xenotransplantation as a tool in reproductive biology and discusses the ethical and potential safety issues regarding ovarian tissue xenotransplantation as a means of recovering fertility.

Changes in Organization of the Endoplasmic Reticulum during Xenopus Oocyte Maturation and ActivationV⃞

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releaseability of Ca2+ by IP3. The clusters dispersed during the Ca2+ wave at activation. Possible relationships of ER structure and Ca2+ regulation are discussed.