El consumo semicrónico moderado de alcohol altera la foliculogénesis y la calidad núcleo-citoplasmática oocitaria, en el ratón / Moderate semi-chronic alcohol consumption alters folliculogenesis and oocyte nuclear-cytoplasmic quality in mice

Resumen El consumo crónico de alcohol es un problema de salud mundial que afecta particularmente a la población femenina. Sin embargo, los efectos de la ingesta semicrónica en cantidades moderadas a bajas en el ovario y el oocito son poco conocidos. En un modelo murino, se administró etanol al 10% en agua de bebida (hembras tratadas) o agua (hembras control) por 15 días, y luego de la superovulación o no (ovulación espontánea), se analizó el ciclo estral y la calidad ovárico-gamética. En las hembras tratadas, la frecuencia y duración del diestro aumentó, y las frecuencias de folículos y cuerpos lúteos disminuyeron vs hembras controles, valores que se restauraron luego de la superovulación. Sin embargo, en las hembras tratadas, la tasa de proliferación celular folicular y el desbalance de la expresión ovárica de VEGF (factor de crecimiento endotelial) persistieron luego de la superovulación. El número de ovocitos ovulados con metafase II anormal, fragmentados y activados partenogenéticamente fue mayor en las hembras tratadas respecto las controles. En conclusión, el consumo semicrónico moderado de alcohol produce anestro, ciclo estral irregular, foliculogénesis deficiente y anomalías núcleo-citoplasmáticas en los oocitos ovulados. Estas alteraciones podrían constituirse en un factor etiológico de pérdida gestacional temprana y desarrollo embrionario anormal luego del consumo de alcohol.

Abstract Chronic alcohol consumption is a global health problem that particularly affects the female population. However, the ef-fects of semi-chronic ethanol intake in low-moderate amounts on the ovary and oocyte are poorly understood. In a mouse model, 10% ethanol was administered in drinking water (treated females) or water (control females) for 15 days, and after superovulation or not (spontaneous ovulation), the estrous cycle and ovarian-gametic quality were analyzed. In treated females, the frequency and duration of the diestrus increased, and the frequencies of follicles and corpus luteum decreased vs control females, values that restored after superovulation. However, in treated females, the follicular cell proliferation rate and the imbalance in ovarian expression of VEGF (endothelial growth factor) persisted after superovulation. The number of ovulated oocytes with abnormal metaphase II, fragmented and parthenogenetically activated was higher in treated females than in control ones. In conclusion, moderate semi-chronic alcohol consumption produces anestrum, irregular estrous cycle, poor folliculogenesis, and nuclear-cytoplasmic abnormalities in ovulated oocytes. These alterations could constitute an etiological factor of early gestational loss and abnormal embryonic development after alcohol consumption.

Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles / Intervalo na substituição do meio de cultivo in vitro afeta a integridade e o desenvolvimento de folículos pré-antrais equinos

The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher's test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.(AU)

A eficiência de um sistema de cultivo está relacionada à elaboração e substituição do meio de cultivo com condições adequadas ao desenvolvimento folicular. Pesquisas recentes sugerem que o meio de cultivo in vitro deve ser substituído após períodos de tempo específicos para várias espécies. No entanto, o intervalo adequado para a troca de meio de cultivo in vitro ainda não foi estabelecido na espécie equina. O objetivo desta investigação foi avaliar o efeito de intervalos de troca média de 24 horas (T24) ou 48 horas (T48) para cultivo de folículos pré-antrais aos 2 ou 6 dias. No final do período de cultivo, os fragmentos foram processados ​​usando histologia clássica. Os folículos pré-antrais equinos foram classificados de acordo com a integridade morfológica e o estágio de desenvolvimento. A análise dos dados foi realizada utilizando o teste de Fisher com um nível de significância de p<0,05. De um total de 399 folículos avaliados, 174 (43,6%) foram folículos primordiais, 225 (56,4%) estavam em desenvolvimento e 63,76% estavam morfologicamente intactos. No cultivo in vitro realizado ao longo de dois dias, não houve diferença significativa em relação à integridade folicular após a substituição do meio (p>0,05). Comparado com a substituição média aos seis dias de cultivo, houve diferença estatisticamente significativa para T24 (68,9%, p<0,05). Portanto, sugerimos alterar o meio para as espécies equinas às 48 horas após o início da cultura, seguindo as subsequentes substituições diárias.(AU)

Improvement in Ovarian Tissue Quality with Supplementation of Antifreeze Protein during Warming of Vitrified Mouse Ovarian Tissue

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.

Morphological indicators of initial reproductive commitment in Mustelus schmitti (Springer 1939) (Chondrichthyes, Triakidae): folliculogenesis and ovarian structure over the life cycle / Indicadores morfológicos de compromisso reprodutivo inicial em Mustelus schmitti (Springer 1939) (Chondrichthyes, Triakidae): folliculogenesis e estrutura ovariana ao longo do ciclo de vida

This work provides information about the sexual commitment and the folliculogenesis of the gatuzo, Mustelus schmitti. A total of 112 females of all maturity stages were fished in the Bahía Blanca estuary, between 2009 and 2010. The oogonia were present throughout the life cycle of the animals. The folliculogenesis follows a pattern similar to other elasmobranchs. The granulosa layer keeps monolayered throughout the folliculogenesis, but with two cell types in the vitellogenic follicle. The zona pellucida forms in the primordial follicles. The thecal system shows a connective inner layer and a glandular outer sheath. The microscopic beginning of the sexual commitment, indicated by the vitello hoarding, takes place in follicles from 500 micrometres, while the macroscopic evidence appears in follicles of 2500-3000 micrometres. The results presented in this study suggest that the fishery pressure may affect a susceptible range of sizes of the species, not previously considered and provides a biological framework for the development of fisheries policy.

Este trabalho provê informações sobre o compromisso sexual e da foliculogênese do gatuzo, Mustelus schmitti. Um total de 112 fêmeas de todas as fases de maturidade foram pescados no estuário Bahía Blanca, entre 2009 e 2010. O oogônias foi presentes durante todo do ciclo de vida dos animais. A foliculogênese segue um padrão semelhante a outros elasmobrânquios. A capa granulosa mantém-se simples durante toda a foliculogénese, mas com dois tipos de células no folículo vitelogênico. A zona pelúcida forma-se nos folículos primordiais. O sistema mostra uma capa tecal interior de tecido conjuntivo e uma bainha exterior glandular. O início microscópico do compromisso sexual, indicado pela acumulação do vitello, realiza-se em folículos de 500 micrómetros, enquanto que a evidência macroscópica aparece em folículos de 2500-3000 micrómetros. Os resultados apresentados neste estudo sugerem que a pressão da pesca pode afetar um amplo intervalo de tamanho das espécies não considerado anteriormente, e fornece uma base biológica para o desenvolvimento de política comum da pesca.

Evaluation of apoptosis as a mechanism of follicular cell atresia in the ovaries of cattle (bos indicus) and buffalo (bubalus bubalis) fetuses

The objective of this study was to investigate the occurrence of apoptosis in the ovaries of cattle and buffalo fetuses between 4 and 8 months old by the terminal deoxynucleotidyltransferase - mediated dUTP nick end labeling (TUNEL) assay . Histological analysis of the ovarian t issue showed that apoptosis occurred at all ages evaluated , presenting a similar pattern among different fetal stages in both species . Within species, secondary follicles displayed a higher (P 0.05) of apoptotic follicular cells among the three follicular classes compared . C omparing resul ts between species, secondary follicles had a higher (P < 0.05) mean number of TUN EL positive cells in bovine fetuses; however , this difference was proportional to the larger number of follicular cells present in secondary follicles in this species . In summary, the TUNEL method was effective for the detection of apoptosis in the support ing cells of ovarian follicles from bovine and buffalo fetuses with apoptosis occurring at similar rates in both species between 4 and 8 months of gestational age. Further studies are needed to better understand the dynamics of apoptosis as a regulator of follicular atresia in fetal ovaries from these species, as well as the potential involvement of the oocyte in this process.

Relevancia de la apoptosis en la reproducción femenina / Relevance of apoptosis in the female reproductive system

La apoptosis es un proceso genéticamente controlado mediante el cual las células inducen su propia muerte. Mensualmente y en forma cíclica, el ovario, el endometrio y la glándula mamaria atraviesan por ciclos de proliferación celular y apoptosis respondiendo a los cambios en la secreción hormonal. Durante el desarrollo embrionario, la apoptosis está implicada en procesos relacionados con la escultura de los diferentes órganos, a través de la eliminación de estructuras innecesarias y con el control de las células defectuosas. Asimismo, la apoptosis juega un papel fundamental en la función ovárica. La reserva folicular se establece durante la vida fetal y luego se va eliminando gradualmente. La apoptosis está involucrada tanto en la muerte celular durante el proceso de reclutamiento del folículo dominante, como en la luteólisis. Durante la pubertad la apoptosis contribuye a la formación del espacio luminal de los ductos terminales de la mama. A su vez, el proceso de involución mamaria luego de la lactancia se caracteriza por una apoptosis masiva de las células epiteliales secretoras. Así como la apoptosis está involucrada en los cambios fisiológicos que ocurren a nivel endometrial, también se ha asociado a la muerte celular programada con procesos patológicos, especialmente en aquellos caracterizados por el incremento en el crecimiento celular como es el caso de la endometriosis. El delicado balance entre la apoptosis y la proliferación celular es fundamental ya que permite que los tejidos puedan responder en forma cíclica a los cambios hormonales fisiológicos y prevenir procesos de transformación neoplásica.

Apoptosis is a genetically controlled form of cell suicide. Due to the cyclic nature of the female reproductive system, the ovary, the endometrium and the mammary gland sustain continuous cycles of cell growth and apoptosis in response to hormonal changes. Apoptotic cell death plays multiple roles during embryonic and organ development. It is involved in sculpturing tissues and serves to delete structures that are no longer required. It is clear that apoptosis plays an active and important role in ovarian physiological functions. Apoptosis plays a major role during folliculogenesis and dominant follicle selection and also plays part in corpus luteum regression. In addition, it has been shown that programmed cell death plays important roles in the mammary gland development and ductal morphogenesis. During puberty, lumen formation is associated with the selective apoptosis of centrally located cells. In turn, postlactational involution of the mammary gland is characterized by the secretory epithelial cells undergoing programmed cell death. Apoptosis has also been associated with physiological, as well as pathological, endometrial processes such as cancer and endometriosis. The delicate balance between apoptosis and cell proliferation is essential in controlling the cyclical growth of the reproductive tissues and plays an important role in the prevention of neoplastic transformation.

The ovary of Lagostomus maximus (Mammalia, Rodentia): an analysis by confocal microscopy

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 µm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species.

Assessment of gonadal follicle size in the invading bivalve Limnoperna fortunei (Mytilidae)

Limnoperna fortunei is an invasive gonochoristic and byssate freshwater bivalve originary from Southeast Asia. It shows great adaptive-reproductive ability, so knowledge of the gonadal cycle is an important factor for the prevention and control of this bioinvasion. This species is highly damaging to natural and human environments. We analyzed the distribution and maturity state of reproductive follicles in the mantle of both male and females. Male results are not shown but, in general, they followed the same pattern as that of females. Routine histological techniques included serial longitudinal sections and transversal sections in three body regions (anterior, middle and psoterior). Oocytes with conspicuous nucleoli were measured on both types of sections to estimate the maturity stage in the different regions. ANOVA indicates that there were no significant differences in maturity ratio between the studied regions, so that a small number of sections would render precise results to assess maturity

Assessment of gonadal follicle size in the invading bivalve Limnoperna fortunei (Mytilidae)

Limnoperna fortunei is an invasive gonochoristic and byssate freshwater bivalve originary from Southeast Asia. It shows great adaptive-reproductive ability, so knowledge of the gonadal cycle is an important factor for the prevention and control of this bioinvasion. This species is highly damaging to natural and human environments. We analyzed the distribution and maturity state of reproductive follicles in the mantle of both male and females. Male results are not shown but, in general, they followed the same pattern as that of females. Routine histological techniques included serial longitudinal sections and transversal sections in three body regions (anterior, middle and psoterior). Oocytes with conspicuous nucleoli were measured on both types of sections to estimate the maturity stage in the different regions. ANOVA indicates that there were no significant differences in maturity ratio between the studied regions, so that a small number of sections would render precise results to assess maturity

Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation

OBJECTIVE: To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozenthawed samples. METHODS: Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed. RESULTS: We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin-stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm3 for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4 percent) when compared with the cryopreserved tissues (70.8 percent for G30 (p<0.001) and 78.4 percent for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation. CONCLUSION: The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30 percent decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.

Soluble Human Leukocyte Antigen G Level in Fluid from Single Dominant Follicle and the Association with Oocyte Competence

PURPOSE: To investigate the direct relationship between the follicular fluid (FF) level of soluble human leukocyte antigen G (HLA-G) and fertilizability of the corresponding oocyte as well as the morphological quality of the corresponding embryo. MATERIALS AND METHODS: Sixty-three patients were stimulated with recombinant FSH combined with gonadotropin-releasing hormone (GnRH) agonist long (n=5) or antagonist protocol (n=58) for standard in vitro fertilization (IVF). At the time oocyte retrieval, follicular fluid was obtained from single dominant follicle in 63 patients, and the level of soluble HLA-G was measured by sandwich enzyme-liked immunosorbent assay (ELISA). Normal fertilization and individual embryo quality were evaluated, and were graded to four categories by morphological criteria (the embryo with symmetrical blastomeres and no fragmentation were assigned as grade A). Good-quality embryo was defined as those with grade A or B. RESULTS: Soluble HLA-G was not detected in 15 FF samples. In the group with positive FF soluble HLA-G (sHLA-G) (n=48), high levels of sHLA-G (>117.758 U/mL) could predict the failure of fertilization with statistical significance {area under the curve (AUC) 0.676, 95% confidence interval (CI) 0.525-0.804}. However, the FF sHLA-G level was not related with the formation of good-quality embryo. CONCLUSION: High level of FF sHLA-G could predict the fertilization failure of the corresponding oocyte, but was not related with the formation of good-quality embryo.

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3 percent), and the highest percent of primary follicles was achieved with IGF-I (57.7 percent). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations

Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production.

Impact of pituitary FSH purification on in vitro early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.

Programmed cell death during ovarian differentiation in queens of Apis mellifera Linné, 1758 (Hymenoptera, Apini)

In this study, we examined various aspects of ovarian development in adult honey bees queens (Apis mellifera). Caged honey bee queens showed an initial programmed germ cell differentiation that was independent of any external or environmental stimulus. In young queens, division of the stem germ cells resulted in cysts of clone cells (cystocytes) that were connected through intercellular bridges and appeared as rosettes. The cystocytes started differentiate shortly before the queens reached sexual maturity (about 5 days old). The oocyte subsequently appeared as a large, stained cell connected to parallel, double rows of smaller cells, or nurse cells. If the queens did not mate, germ cell differentiation was “switched off”, and development of the ovary was interrupted in an intermediate developmental stage, without follicular cell organization. Therefore, in such queens, there were no pevitellogenic follicles. This finding could explain why wvirgin queens rarely lay eggs and why after fecundation they require several days to start laying. The absence of previtellogenic follicles may also indicate that some stimulus is required for continuation of the vitellogenesis and ovary development.

Histochemical study of the extracellular matrix components in the follicular wall of induced polycystic ovaries

Numerous models have been developed to study polycystic ovarian syndrome in rats. In the present study, the syndrome was induced by exposure to constant light. The histological structure and differential distribution of extracellular matrix (ECM) fibers as well as the glycosaminoglycans (GAGs) content and composition of the ovarian follicular wall of rats with polycystic syndrome were evaluated. Histochemical differences were observed in the graunlosa and theca externa of follicular cysts when compared to normal preovulatory follicles. The colagen content of the theca externa of follicular cysts, quantified by the picrosirius method, was higher than in the controls. The neural carbohydrate and acidic GAC levels were lower in the granulosa and higher in the theca externa of cyst follicles than in control ovaries. Histomorphometrically, the follicular diameter was both a convenient and appropriate measurement for describing the cyst status; there were no differences in the thickness of each follicular layer. In conclusion, differences in the components of ECM were observed in the follicular wall of ovarian cysts compared eith normal preovulatory follicles. Howere, sinde these changes did not occur uniformly in all layers of the follicular wall, their role in cyst development remains to be established.

Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans

The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu)

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.

Cystogenesis of antral follicles induced by dehydroepiandrosterone (DHEA) stimulates mast cell proliferation and maturation in the house rat (Rattus rattus) ovary.

Mast cell dynamics has been studied in relation to cystogenesis of ovarian follicles in the house rat. Immature rats were injected (s.c.) daily with DHEA (6.0 mg/100 g body weight) and were sacrificed on the day 8, 16 and 24 of the start of treatment. Ovarian sections of the treated rats had majority of the antral follicles undergoing atresia or in early stages of cystogenesis. Completely developed cysts were evident from the ovarian surface after 24 days of daily treatment. Treatment for 8 days resulted in significant increase in the number of alcian blue-positive ovarian mast cells. Ovaries after 16 days of DHEA treatment showed no marked change with regard to the number of total mast cells per unit area and staining characteristics. However, a significant rise in ovarian mast cell counts was recorded after 24 days of treatment and most of the cells contained safranin-positive red granules. This increase was attributed due to the increase in their number in medulla and stroma around the cystic follicles.