Comparison of age-related anti Müllerian hormone, inhibin-b levels and follicle reserve in rat ovarium / Comparación de la hormona anti-mülleriana relacionada con la edad, los niveles de inhibina-b y la reserva folicular en el ovario de rata

SUMMARY: Anti-Müllerian hormone (AMH) and Inhibin B (INHB) in the glycoprotein structure are members of the transforming growth factor β family and expressed by granulosa cells from puberty. AMH is a factor that increases the life span of small developing follicles. For this reason, it is widely used to determine the ovarian reserve and age. Inhibin-B secreted from granulosa cells plays a role in regulation of the Follicle Stimulating Factor (FSH) and determination of the follicle diameter. There are few studies on the effect of these two age-related hormones on ovarian histology in rats. In this study, AMH and INHB expression in ovarian tissues of female rats of different age groups, their relationship with ovarian structure and folliculogenesis were examined histologically and biochemically. Wistar Albino rats were used in the study and a total of 3 groups were formed. The ovaries of rats in the pre-oestrous period were collected, and follicle count was performed on tissue sections in batches. Expression of AMH in the follicles was identified immunohistochemically. In serum, AMH and INHB levels were assessed by ELISA method and their significance was evaluated statistically. Results from light microscopic examination determined that AMH was expressed from the granulosa cells of developing follicles. INHB expression during the prepubertal period and AMH had a protective effect on the ovarian reserve and the number of developing follicles, respectively.

RESUMEN: La hormona antimülleriana (AMH) y la inhibina B (INHB) en la estructura de la glicoproteína son miembros de la familia del factor de crecimiento transformante β y se expresan en las células de la granulosa desde la pubertad. La AMH es un factor que aumenta la vida útil de los pequeños folículos en desarrollo. Por este motivo, se utiliza frecuentemente para determinar la reserva ovárica y la edad. La inhibina B secretada por las células de la granulosa tiene un rol en la regulación del factor estimulante de (FSH) y en la determinación del diámetro del folículo. Hay pocos estudios sobre el efecto de estas dos hormonas relacionadas con la edad en la histología ovárica en ratas. Se examinaron histológica y bioquímicamente la expresión de AMH e INHB en tejidos ováricos de ratas hembras de diferentes grupos de edad, su relación con la estructura ovárica y la foliculogénesis. Se utilizaron ratas Wistar Albino en el estudio y se formaron 3 grupos. En los ovarios de ratas en el período preestro se realizó el recuento de folículos en secciones de tejido. La expresión de AMH en los folículos se identificó inmunohistoquímicamente. En suero, los niveles de AMH e INHB se evaluaron mediante el método ELISA y su importancia se evaluó estadísticamente. Los resultados del examen con microscopio óptico determinaron que la AMH se expresaba a partir de las células de la granulosa de los folículos en desarrollo. La expresión de INHB durante el período prepuberal y AMH tuvo un efecto protector sobre la reserva ovárica y el número de folículos en desarrollo, respectivamente.

A physiologic overview of the organ-specific transcriptome of the cattle tick Rhipicephalus microplus.

To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.

Developmental programming: intrauterine caloric restriction promotes upregulation of mitochondrial sirtuin with mild effects on oxidative parameters in the ovaries and testes of offspring.

According to the developmental origins of health and disease (DOHaD) hypothesis, changes in the maternal environment are known to reprogram the metabolic response of offspring. Known for its redox modulation, caloric restriction extends the lifespan of some species, which contributes to diminished cellular damage. Little is known about the effects of gestational caloric restriction, in terms of antioxidant parameters and molecular mechanisms of action, on the reproductive organs of offspring. This study assessed the effects of moderate (20%) caloric restriction on redox status parameters, molecular expression of sirtuin (SIRT) 1 and SIRT3 and histopathological markers in the ovaries and testes of adult rats that were subjected to gestational caloric restriction. Although enzyme activity was increased, ovaries from female pups contained high levels of oxidants, whereas testes from male pups had decreased antioxidant enzyme defences, as evidenced by diminished glyoxalase I activity and reduced glutathione content. Expression of SIRT3, a deacetylase enzyme related to cellular bioenergetics, was increased in both ovaries and testes. Previous studies have suggested that, in ovaries, diminished antioxidant metabolism can lead to premature ovarian failure. Unfortunately, there is little information regarding the redox profile in the testis. This study is the first to assess the redox network in both ovaries and testes, suggesting that, although intrauterine caloric restriction improves molecular mechanisms, it has a negative effect on the antioxidant network and redox status of reproductive organs of young adult rats.

Stranded false killer whales, Pseudorca crassidens, in Southern South America reveal potentially dangerous silver concentrations.

Silver (Ag) is a non-essential metal known to bioaccumulate in aquatic organisms. We determined Ag concentrations in five false killer whales stranded in South America. Silver concentrations (in dry weight basis) range as 6.62-10.78 µg g-1 in liver, 0.008-7.41 µg g-1 in spleen, 0.004-5.71 µg g-1 in testis, 0.757-1.69 µg g-1 in kidney, 0.011-0.078 µg g-1 in lung and < 0.01-0.038 µg g-1 in muscle, whereas in the single samples of uterus and ovary were 0.051 and 0.023 µg g-1; respectively. Overall, Ag concentration in liver and kidney exceeded the cetacean toxic thresholds, proposed as "unhealthy concentrations" and "critically dangerous" in liver and kidney. These results warrant further eco-toxicological studies, to examine biological effects of elevated silver levels for individuals and to assess the species' conservation status with respect to marine pollution.

Immunohistochemical localization of 3ß-Hydroxysteroid dehydrogenase and progesterone receptors in the ovary and placenta during gestation of the placentotrophic lizard Mabuya sp (Squamata: Scincidae).

In squamates, progesterone (P) plays a key role in the inhibition of uterine mobility during egg retention in oviparous species, and during gestation in viviparous species. The corpus luteum (CL) is the main organ responsible for the production of P; however, in some species, the CL degenerates early and the P needed for gestation maintenance should be produced in other tissues. Mabuya sp (Scincidae) is a viviparous lizard with a prolonged gestation, it produces microlecithal eggs and, consequently, has an obligate placentotrophy related with a highly complex placenta. Its CL degenerates at early stages of gestation and therefore, other sources of P should exist. The aim of this study was to determine and localize by immunohistochemistry the production of P by detection of the enzyme 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) and P receptors (PR) during gestation in the ovary and placenta of Mabuya sp. Positive and negative control sections were used. The ovary of this species localizes 3ß-HSD and PR in the same tissues. The CL of the ovaries of females at early stages of gestation were positive for both molecules, whereas they did not localize from mid gestation to the end of pregnancy. Previtellogenic and vitellogenic follicles labelled for both molecules in the follicular epithelium and thecae. The placenta of Mabuya sp. demonstrated the potential for P production from mid gestation to the end of gestation in the uterine and chorionic tissues. PR were located in the uterine tissues throughout gestation, with a decrease towards its completion. Western blot analysis confirmed the presence of 3ß-HSD mainly in the ovary of early pregnant females and in the placental tissues at mid gestation stages. Therefore, the chorioallantoic placenta of Mabuya sp. has an endocrine function producing the P needed for gestation and replacing the CL from mid gestation to the end of pregnancy.

Imunolocalização de angiotensina-(1-7), angiotensina II e Enzima Conversora de Angiotensina tipo II em ovário de cabra / Immunolocalization of angiotensin-(1-7), angiotensin II and Angiotensin Converting Enzyme II in goat ovary

There is increasing evidence the participation of ovarian renin-angiotensin system in importantreproductive processes. This study aimed to investigate the presence and location of Ang II, Ang-(1-7) and ACE2in goat ovaries. Ten ovaries from goats killed in slaughterhouse were collected, washed in buffered PBS,perfused with protease inhibitor solution and processed for histology standard protocol. The search of peptideswas performed using the avidin–biotin–peroxidase method. A strong immunoreactivity for Ang II in theca cellsof antral follicles and corpora lutea was observed. Antral follicles (theca cells), corpora lutea and oocytecytoplasm in early antral follicles exhibited strong immunoreactivity for Ang-(1-7). There was strongimmunoreactivity for ACE2 in the cytoplasm of luteal cells and theca cells of antral follicles. For the first time,the presence and location of Ang II, Ang-(1-7) and ACE2 are reported in goat ovary, can regulate folliculardevelopment, oocyte maturation and corpus luteum development.(AU)

Imunolocalização de angiotensina-(1-7), angiotensina II e Enzima Conversora de Angiotensina tipo II em ovário de cabra / Immunolocalization of angiotensin-(1-7), angiotensin II and Angiotensin Converting Enzyme II in goat ovary

There is increasing evidence the participation of ovarian renin-angiotensin system in importantreproductive processes. This study aimed to investigate the presence and location of Ang II, Ang-(1-7) and ACE2in goat ovaries. Ten ovaries from goats killed in slaughterhouse were collected, washed in buffered PBS,perfused with protease inhibitor solution and processed for histology standard protocol. The search of peptideswas performed using the avidin–biotin–peroxidase method. A strong immunoreactivity for Ang II in theca cellsof antral follicles and corpora lutea was observed. Antral follicles (theca cells), corpora lutea and oocytecytoplasm in early antral follicles exhibited strong immunoreactivity for Ang-(1-7). There was strongimmunoreactivity for ACE2 in the cytoplasm of luteal cells and theca cells of antral follicles. For the first time,the presence and location of Ang II, Ang-(1-7) and ACE2 are reported in goat ovary, can regulate folliculardevelopment, oocyte maturation and corpus luteum development.

Characterization of the ovary fatty acids composition of Rhamdia quelen (Quoy & Gaimard) (Teleostei: Siluriformes), throughout their reproductive cycle

Knowledge about gonad fatty acid composition is important for broodstock diet formulation. This study characterized ovary fatty acid composition of wild female jundiá catfish (Rhamdia quelen) in their different gonadal maturation stages. Female jundiá (n = 36, average weight= 383.8 + 208.8 g) were captured in the rio Uruguay, comprising all seasons. Ovaries were extracted and classified according to their gonadal maturation stage. Gonad-somatic ratio varied significantly among seasons, being higher in spring (3.7), followed by summer (2.2), winter (0.9) and autumn (0.6). Main fatty acids groups detected were: saturated (SFA= 35.5%), monounsaturated (MUFA= 28.1%) and polyunsaturated fatty acids (PUFA= 33.5%). Over the four seasons, palmitic acid was recorded in large quantities, followed by docosahexaenoic acid (DHA) and arachidonic acid (ARA). ARA was present in higher concentrations in immature or maturing ovaries, and its content decreased along the maturation process. Conversely, DHA and eicosapentaenoic acid (EPA) contents increased during maturation. Such variation resulted in an increase in EPA/ARA and DHA/ARA ratios in mature gonads, which can be important for successful breeding. Such findings suggest that jundiá broodstock diets should contain lipids that provide long chain polyunsaturated fatty acids from both the n-3 and n-6 series to ensure gonadal maturation completion.

O conhecimento da composição de ácidos graxos da gônada, como dos fatores que a influenciam são importantes na formulação de dietas para reprodutores de qualquer espécie. O presente estudo caracteriza a composição de ácidos graxos da gônada de fêmeas de jundiá (Rhamdia quelen) selvagens em seus diferentes estágios de maturação gonadal. Fêmeas de jundiá (n = 36) com peso médio de 383,8 + 208,8 g foram capturadas no alto rio Uruguai, ao longo de um ano, abrangendo as quatro estações. As gônadas foram extraídas, classificadas e posteriormente sua composição de ácidos graxos foi determinada. A relação gônado-somática variou significativamente entre as estações, sendo maior na primavera (3,7), seguida de verão (2,2), inverno (0,9) e no outono (0,6). Os principais grupos de ácidos graxos detectados foram: saturados (35,5 + 3,5%), monoinsaturados (28,1 + 4,3%) e ácidos graxos poli-insaturados (33,5 + 3,0%), com aproximadamente 30% para cada grupo. Ao longo das quatro estações, o ácido palmítico foi registrado em grandes quantidades, seguido do ácido docosahexaenóico (DHA) e ácido araquidônico (ARA). O ácido araquidônico estava presente em concentrações mais elevadas nas gônadas imaturas que nas gônadas maturas, e seu conteúdo diminuído ao longo do processo de maturação. Inversamente, o conteúdo de DHA e ácido eicosapentaenoico (EPA) aumentaram durante o processo de maturação. Tal variação resultou num aumento das relações EPA/ARA e DHA/ARA nas gônadas maturas, fato que pode ser importante para o sucesso da reprodução no jundiá. Tais resultados sugerem que dietas para reprodutores da espécie devem conter fontes de lipídios que proporcionem ácidos graxos de cadeia longa de ambas series (n-3 e n-6) para assegurar uma correta maturação final da gônada.

Characterization of the ovary fatty acids composition of Rhamdia quelen (Quoy & Gaimard) (Teleostei: Siluriformes), throughout their reproductive cycle

Knowledge about gonad fatty acid composition is important for broodstock diet formulation. This study characterized ovary fatty acid composition of wild female jundiá catfish (Rhamdia quelen) in their different gonadal maturation stages. Female jundiá (n = 36, average weight= 383.8 + 208.8 g) were captured in the rio Uruguay, comprising all seasons. Ovaries were extracted and classified according to their gonadal maturation stage. Gonad-somatic ratio varied significantly among seasons, being higher in spring (3.7), followed by summer (2.2), winter (0.9) and autumn (0.6). Main fatty acids groups detected were: saturated (SFA= 35.5%), monounsaturated (MUFA= 28.1%) and polyunsaturated fatty acids (PUFA= 33.5%). Over the four seasons, palmitic acid was recorded in large quantities, followed by docosahexaenoic acid (DHA) and arachidonic acid (ARA). ARA was present in higher concentrations in immature or maturing ovaries, and its content decreased along the maturation process. Conversely, DHA and eicosapentaenoic acid (EPA) contents increased during maturation. Such variation resulted in an increase in EPA/ARA and DHA/ARA ratios in mature gonads, which can be important for successful breeding. Such findings suggest that jundiá broodstock diets should contain lipids that provide long chain polyunsaturated fatty acids from both the n-3 and n-6 series to ensure gonadal maturation completion.(AU)

O conhecimento da composição de ácidos graxos da gônada, como dos fatores que a influenciam são importantes na formulação de dietas para reprodutores de qualquer espécie. O presente estudo caracteriza a composição de ácidos graxos da gônada de fêmeas de jundiá (Rhamdia quelen) selvagens em seus diferentes estágios de maturação gonadal. Fêmeas de jundiá (n = 36) com peso médio de 383,8 + 208,8 g foram capturadas no alto rio Uruguai, ao longo de um ano, abrangendo as quatro estações. As gônadas foram extraídas, classificadas e posteriormente sua composição de ácidos graxos foi determinada. A relação gônado-somática variou significativamente entre as estações, sendo maior na primavera (3,7), seguida de verão (2,2), inverno (0,9) e no outono (0,6). Os principais grupos de ácidos graxos detectados foram: saturados (35,5 + 3,5%), monoinsaturados (28,1 + 4,3%) e ácidos graxos poli-insaturados (33,5 + 3,0%), com aproximadamente 30% para cada grupo. Ao longo das quatro estações, o ácido palmítico foi registrado em grandes quantidades, seguido do ácido docosahexaenóico (DHA) e ácido araquidônico (ARA). O ácido araquidônico estava presente em concentrações mais elevadas nas gônadas imaturas que nas gônadas maturas, e seu conteúdo diminuído ao longo do processo de maturação. Inversamente, o conteúdo de DHA e ácido eicosapentaenoico (EPA) aumentaram durante o processo de maturação. Tal variação resultou num aumento das relações EPA/ARA e DHA/ARA nas gônadas maturas, fato que pode ser importante para o sucesso da reprodução no jundiá. Tais resultados sugerem que dietas para reprodutores da espécie devem conter fontes de lipídios que proporcionem ácidos graxos de cadeia longa de ambas series (n-3 e n-6) para assegurar uma correta maturação final da gônada.(AU)

Immunohistochemical localization of fibroblast growth factor-2 in the sheep ovary and its effects on pre-antral follicle apoptosis and development in vitro.

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF-2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10 ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.

Comparing two different superovulation protocols on ovarian activity and fecal glucocorticoid levels in the brown brocket deer (Mazama gouazoubira).

BACKGROUND: Stress is a limiting factor in assisted reproduction in wild animals maintained in captivity. However, the knowledge of assisted reproduction techniques for wild animals is useful for future in situ and ex situ conservation programs. Thus, this study evaluated the ovulation rate, presence of functional corpora lutea and fecal glucocorticoid levels following treatments promoting superovulation in captive brown brocket deer. METHODS: The crossover design used six hinds, allocated to two groups (n=6): eCG Treatment, CIDR for 8 days, followed by 0.25 mg of EB on day 0, 700 IU of eCG on day 4 following device insertion and 265 mug of PGF2alfa on day 8; and FSH Treatment, CIDR for 7.5 days, followed by 0.25 mg of EB on day 0, 130 mg of FSH in 8 equal doses and 265 mug of PGF2alfa on day 7.5. Induced adrenal activity and treatment efficacy were evaluated by corpora lutea (CL) counts and fecal glucocorticoid and progestin concentration (ng/g feces) analyses for five different phases: Pre, two days before treatment; Early, first four days of treatment; Late, last four days of treatment; Total, entire treatment period; and Post, five days posttreatment. RESULTS: eCG Treatment resulted in the highest number of CL (P lower than 0.05). There was no significant difference for fecal glucocorticoid concentrations in five different time periods between the treatments; however Pre fecal glucocorticoid concentrations (90.06+/-19.64) were significantly different from Late (200.76+/-26.39) within FSH Treatment. The mean fecal progestin concentration and mean ovulation rate were higher in eCG Treatment (4293.69+/-769.47, 7.0+/-1.8) than in FSH Treatment (1571.26+/-240.28, 2.6+/-0.8) (P lower than or equal to 0.05). CONCLUSIONS: Although the eCG Treatment induced a good superovulatory response, with the formation of functional corpora lutea, we cannot yet affirm that we have established a suitable protocol for induction of SOV in the species M. gouazoubira because approximately 65% of the deer showed premature regression of the corpora lutea. Moreover, multiple FSH applications in FSH Treatment resulted in a low ovulation rate and induced an increase in fecal glucocorticoid levels.

Quantification of healthy and atretic germ cells and follicles in the developing and post-natal ovary of the South American plains vizcacha, Lagostomus maximus: evidence of continuous rise of the germinal reserve.

The female germ line in mammals is subjected to massive cell death that eliminates 60-85% of the germinal reserve by birth and continues from birth to adulthood until the exhaustion of the germinal pool. Germ cell demise occurs mainly through apoptosis by means of a biased expression in favour of pro-apoptotic members of the BCL2 gene family. By contrast, the South American plains vizcacha, Lagostomus maximus, exhibits sustained expression of the anti-apoptotic BCL2 gene throughout gestation and a low incidence of germ cell apoptosis. This led to the proposal that, in the absence of death mechanisms other than apoptosis, the female germ line should increase continuously from foetal life until after birth. In this study, we quantified all healthy germ cells and follicles in the ovaries of L. maximus from early foetal life to day 60 after birth using unbiased stereological methods and detected apoptosis by labelling with TUNEL assay. The healthy germ cell population increased continuously from early-developing ovary reaching a 50 times higher population number by the end of gestation. TUNEL-positive germ cells were <0.5% of the germ cell number, except at mid-gestation (3.62%). Mitotic proliferation, entrance into prophase I stage and primordial follicle formation occurred as overlapping processes from early pregnancy to birth. Germ cell number remained constant in early post-natal life, but a remnant population of non-follicular VASA- and PCNA-positive germ cells still persisted at post-natal day 60. L. maximus is the first mammal so far described in which female germ line develops in the absence of constitutive massive germ cell elimination.

Serum levels of LH, FSH, estradiol and progesterone in female rats experimentally infected by Trypanosoma evansi.

The goal of this study was to evaluate reproductive hormones in sera samples of female rats experimentally infected by Trypanosoma evansi during different phases of the estrous cycle. For that, 64 animals were divided into two groups: 24 rats for the control group (uninfected), and 40 animals were infected by T. evansi. These groups were divided into subgroups according to the time of infection (days 5 and 15 post-infection; PI) and the phase of the estrous cycle (proestrus, estrus, metestrus and diestrus). Serum was collected at days 5 and 15 PI and the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone and estradiol were assessed by enzyme immunoassay technique. The concentration of nitrite/nitrate (NOx), advanced oxidation protein products (AOPP), and thiobarbituric acid reactive substances (TBARS) were measured in ovaries and uteruses in these same periods. Infected females showed significant decrease (P<0.05) of LH, FSH, estradiol and progesterone in different periods and phases of the estrous cycle when compared to uninfected rats. In addition, it was observed an increase in the concentration of NOx, AOPP, and TBARS in the ovaries, which is indicative of cell damage. Therefore, our experimental study showed that T. evansi infection in female rats may cause changes in LH, FSH, estradiol, and progesterone levels regardless of the time of infection or phase of the estrous cycle.

Cytochemistry of fat body trophocytes and ovaries of workers and queens of Melipona quadrifasciata anthidioides (Hymenoptera: Apidae: Meliponini) during vitellogenesis.

The fat body (FB) of insects is where yolk proteins are synthesized. Therefore, relationships between the FB and oogenesis were studied in nurse workers, virgins, and physogastric queens of Melipona quadrifasciata anthidioides, a stingless bee in which the workers produce and lay eggs while provisioning brood cells. The relationships between FB and oogenesis, as well as the routes of materials from hemolymph to the oocytes, were studied through the cytochemical detection of lipids by osmium imidazole (OI), carbohydrates by ruthenium red (RR) and basic proteins by ammoniacal silver (AS). The results show differences in the presence of the studied materials in FB trophocytes and ovary of the classes of females studied and oogenesis phases. Material that tested positive for the treatments was detected among the classes of individuals studied in both, trophocytes and oocytes, and in the route of those materials from hemolymph to the oocytes. The differences found among the individual classes indicate relationships with the nutrition and adaptation to the parsimonious use of nutrients in the metabolism of reproduction.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle.

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

La langosta de agua dulce Cherax quadricarinatus es una especie tropical de gran interés para la acuicultura. Se midió la concentración de vitelogenina (Vg) en hemolinfa, ovario y hepatopáncreas de hembras adultas de esta especie, por medio de ELISA. Las mediciones fueron hechas en los cuatro períodos del ciclo reproductivo: pre-reproductivo, reproductivo medio, reproductivo tardío y post-reproductivo. Se detectaron variaciones significativas en los niveles de Vg tanto en hemolinfa como en hepatopáncreas, se observó el mayor valor durante el período reproductivo medio. Además, tales variaciones se correlacionaron positivamente con el índice hepatosomático. Se observó además una correlación positiva de los niveles de Vg entre hepatopáncreas y ovario. Estos resultados apoyan evidencias previas sobre el papel central del hepatopáncreas como sitio de síntesis de Vg, en esta especie, y también enfatizan la importancia de la hemolinfa para el transporte de la Vg del hepatopáncreas al ovario. Para propósitos de acuicultura, la medición de Vg en hemolinfa podría ser utilizada como un método no lesivo, con el fin de constatar la actividad reproductiva de hembras de C. quadricarinatus.

Gene expression and immunolocalization of fibroblast growth factor 2 in the ovary and its effect on the in vitro culture of caprine preantral ovarian follicles.

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.

Cell proliferation and survival mechanisms underlying the abnormal persistence of follicular cysts in bovines with cystic ovarian disease induced by ACTH.

Cystic ovarian disease (COD) is an important cause of infertility that affects cattle. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, the objective in the present study was to evaluate apoptosis and proliferation in induced ovarian cystic follicles in cows to investigate the follicular persistence. Stage of estrous cycle was synchronized in 10 heifers and 5 were then subjected to the induction of COD by administration of ACTH. After the ovariectomy number of in situ apoptotic cells by TUNEL assay, active caspase-3, FAS/FASLG and members of the BCL2 family were compared by immunohistochemistry and multiplex PCR and cell proliferation by evaluation of Ki-67 protein and cyclin D1 and E mRNA. Significantly (p<0.05) lesser proliferative and apoptotic rates were found in cystic follicles from cows with COD compared with those with regular cycles. The relatively minimal proliferation found by immunohistochemistry with Ki-67 marker were confirmed by the gene expression of cyclin D1 and E. Lesser apoptotic rates were associated with decreased amounts of apoptotic-related proteins BAX, FASLG and caspase-3 as well as the in situ apoptosis detected by TUNEL assay, and increased amounts of the anti-apoptotic survival factor cellular BCL2 in the cystic follicles of the COD group. The BAX/BCL2 gene expression profile confirmed the immunohistochemical findings. Results from the present study indicate that cellular proliferation and apoptosis are altered in cystic follicles of cattle. The present study provides new insights into the molecular mechanisms underlying the aberrant persistence of follicular cysts and related diseases.

Obesity induced by high-fat diet promotes insulin resistance in the ovary.

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNFalpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity.

Sulfated alpha-L-galactans from the sea urchin ovary: selective 6-desulfation as eggs are spawned.

The sea urchin eggs are surrounded by a jelly coat, which contains sulfated polysaccharides with unique structures. These molecules are responsible for inducing the species-specific acrosome reaction, an obligatory event for the binding of sperm and fusion with the egg. The mechanism of biosynthesis of these sulfated polysaccharides is virtually unknown. The egg jelly of the sea urchin Echinometra lucunter contains a simple 2-sulfated, 3-linked alpha-L-galactan. Here, we pulse labeled the sea urchin ovary in vitro with (35)S-sulfate to follow the biosynthesis of the sulfated alpha-L-galactan. We found that the ovary contains a 2,6-disulfated, 3-linked alpha-L-galactan, which incorporates (35)S-sulfate more avidly than the 2-sulfated isoform. The 2,6-disulfated alpha-L-galactan was purified by anion exchange chromatography, analyzed by electrophoresis and characterized by 1D and 2D nuclear magnetic resonance spectra. We also investigated the location of the sulfated polysaccharides on the oocytes using histochemical procedures. The stain revealed high amounts of sulfated polysaccharide in mature oocytes and accessory cells. The amount of intracellular sulfated polysaccharides decreased as oocytes are spawned. We speculate that 2,6-disulfated galactan is initially synthesized in the ovary and that 6-sulfate ester is removed when the polysaccharide is secreted into the egg jelly. Similar events related to remodeling of sulfated polysaccharides have been reported in other biological systems.