Impact of Heat Stress on Oocyte Developmental Competence and Pre-Implantation Embryo Viability in Cattle.

Rectal and vaginal temperatures are utilised in both in vivo and in vitro models to study the effects of heat stress on oocyte competence and embryo viability in cattle. However, uterine temperature increases by only 0.5 °C in heat-stressed cows, significantly lower than simulated increases in in vitro models. Temperature variations within oviducts and ovarian follicles during heat stress are poorly understood or unavailable, and evidence is lacking that oocytes and pre-implantation embryos experience mild (40 °C) or severe (41 °C) heat stress inside the ovarian follicle and the oviduct and uterus, respectively. Gathering detailed temperature data from the reproductive tract and follicles is crucial to accurately assess oocyte competence and embryo viability under realistic heat stress conditions. Potential harm from heat stress on oocytes and embryos may result from reduced nutrient availability (e.g., diminished blood flow to the reproductive tract) or other unidentified mechanisms affecting tissue function rather than direct thermal effects. Refining in vivo stress models in cattle is essential to accurately identify animals truly experiencing heat stress, rather than assuming heat stress exposure as done in most studies. This will improve model reliability and aid in the selection of heat-tolerant animals.

Manejo del Síndrome de Hiperestimulación Ovárica / Management of Ovarian Hyperstimulation Syndrome

INTRODUCCIÓN: El síndrome de hiperestimulación ovárica es una respuesta exagerada del ovario a los tratamientos hormonales para estimular la formación de óvulos. OBJETIVO: Describir el caso clínico de una mujer con síndrome de hiperestimulación ovárica; revisar el abordaje, manejo, tratamiento y cómo prevenirlo. CASO CLÍNICO: Paciente femenina de 37 años, multigesta, en tratamiento con metformina por Síndrome de ovario poliquístico , que presenta infertilidad secundaria a factor tubárico, que desarrolló un cuadro moderado de síndrome de hiperestimulación ovárica como consecuencia de la aplicación de las técnicas de fertilización in vitro (Folitropina alfa humana recombinante (GONAL-F®) y Cetrolerelix (CETROTIDE®); al cuarto día del procedimiento de aspiración folicular presenta dolor pélvico intenso, disuria, deposiciones diarreicas, ecografía abdominal y vaginal evidencia líquido libre en cavidad alrededor de 1000cc, además de ovarios tanto derecho e izquierdo con volumen de 102 mL y 189 mL respectivamente. Paciente es ingresada para realizar tratamiento hidratación parenteral, Enoxaparina 40mg subcutánea, Cabergolina 0.5mg vía oral, alta a las 72 horas. DISCUSIÓN: Las claves para la prevención del síndrome de hiperestimulación ovárica son la experiencia con la terapia de inducción de la ovulación y el reconocimiento de los factores de riesgo para el síndrome de hiperestimulación ovárica. Los regímenes de inducción de la ovulación deberían ser altamente individualizados, monitorizados cuidadosamente y usando dosis y duración mínimas del tratamiento con gonadotropinas para conseguir la meta terapéutica. CONCLUSIONES: El síndrome de hiperestimulación ovárica constituye la complicación más temida durante el uso de inductores de la ovulación; el conocimiento de factores de riesgo, puede prevenir o evitar que llegue a ser de un caso severo, lo cual puede causar mayor morbilidad o hasta mortalidad. La vitrificación se convierte en la técnica que permite prevenir el síndrome de hiperestimulación ovárica, junto con esta técnica hay 2 alternativas: la inducción con análogo de la hormona liberadora de gonadotropina o el uso de agonistas dopaminérgicos.

INTRODUCTION: Ovarian hyperstimulation syndrome is an exaggerated response of the ovary to hormonal treatments to stimulate egg formation. OBJECTIVE: To describe the clinical case of a woman with ovarian hyperstimulation syndrome; to review the approach, management, treatment and how to prevent it. CLINICAL CASE: 37-year-old female patient, multigestation, under treatment with metformin for polycystic ovary syndrome, presenting infertility secondary to tubal factor, who developed a moderate picture of ovarian hyperstimulation syndrome as a consequence of the application of in vitro fertilization techniques (recombinant human follitropin alfa (GONAL-F®) and Cetrolerelix (CETROTIDE®); On the fourth day of the follicular aspiration procedure she presents intense pelvic pain, dysuria, diarrheic stools, abdominal and vaginal ultrasound shows free fluid in the cavity of about 1000cc, in addition to right and left ovaries with a volume of 102 mL and 189 mL respectively. Patient was admitted for parenteral hydration treatment, Enoxaparin 40mg subcutaneous, Cabergoline 0.5mg orally, discharged after 72 hours. DISCUSSION: The keys to prevention of ovarian hyperstimulation syndrome are experience with ovulation induction therapy and recognition of risk factors for ovarian hyperstimulation syndrome. Ovulation induction regimens should be highly individualized, carefully monitored, and using minimal doses and duration of gonadotropin therapy to achieve the therapeutic goal. CONCLUSIONS: Ovarian hyperstimulation syndrome constitutes the most feared complication during the use of ovulation inducers; knowledge of risk factors, may prevent or avoid it from becoming a severe case, which may cause increased morbidity or even mortality. Vitrification becomes the technique that allows preventing ovarian hyperstimulation syndrome, along with this technique there are 2 alternatives: induction with gonadotropin-releasing hormone analog or the use of dopaminergic agonists.

Anatomic nomenclature and 3-dimensional regional model of the human ovary: call for a new paradigm.

The ovaries are the female gonads that are crucial for reproduction, steroid production, and overall health. Historically, the ovary was broadly divided into regions defined as the cortex, medulla, and hilum. This current nomenclature lacks specificity and fails to consider the significant anatomic variations in the ovary. Recent technological advances in imaging modalities and high-resolution omic analyses have brought about the need for revision of the existing definitions, which will facilitate the integration of generated data and enable the characterization of organ subanatomy and function at the cellular level. The creation of these high-resolution multimodal maps of the ovary will enhance collaboration and communication among disciplines and between clinicians and researchers. Beginning in March 2021, the Pediatric and Adolescent Gynecology Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development invited subject-matter experts to participate in a series of workshops and meetings to standardize ovarian nomenclature and define the organ's features. The goal was to develop a spatially defined and semantically consistent terminology of the ovary to support collaborative, team science-based endeavors aimed at generating reference atlases of the human ovary. The group recommended a standardized, 3-dimensional description of the ovary and an ontological approach to the subanatomy of the ovary and definition of follicles. This new greater precision in nomenclature and mapping will better reflect the ovary's heterogeneous composition and function, support the standardization of tissue collection, facilitate functional analyses, and enable clinical and research collaborations. The conceptualization process and outcomes of the effort, which spanned the better part of 2021 and early 2022, are introduced in this article. The institute and the workshop participants encourage researchers and clinicians to adopt the new systems in their everyday work to advance the overarching goal of improving human reproductive health.

Protective effect of gallic acid on doxorubicin-induced ovarian toxicity in mouse.

The aims of the present study were to evaluate the protective effects of gallic acid against doxorubicin-induced ovarian toxicity in mice, and to verify the possible involvement of PI3K and mTOR signaling pathway members (PTEN, Akt, FOXO3a and rpS6) in the gallic acid protective actions. Mice were pretreated with NaCl (0.15 M, p.o.) (control and doxorubicin groups) or gallic acid (50, 100 or 200 mg/kg body weight, p.o.) once daily, for 5 days, and on the third day of treatment, after 1 h of treatment administration, the mice received saline solution (i.p.) (control group) or doxorubicin (10 mg/kg of body weight, i.p.). Next, the ovaries were harvested for histological (follicular morphology and activation), fluorescence (GSH and mitochondrial activity), and immunohistochemical (PCNA, cleaved caspase-3, TNF-α, p-PTEN, Akt, p-Akt, p-rpS6 and p-FOXO3a) analyses. The results showed that cotreatment with 50 mg/kg gallic acid plus doxorubicin preserved the percentage of normal follicles and cell proliferation, reduced the percentage of cleaved caspase-3 follicles, prevented inflammation, and increased GSH concentrations and mitochondrial activity compared to doxorubicin treatment alone. Furthermore, cotreatment 50 mg/kg gallic acid plus doxorrubicin increased expression of Akt, p-Akt, p-rpS6 and p-FOXO3a compared to the doxorubicin alone. In conclusion, 50 mg/kg gallic acid protects the mouse ovary against doxorubicin-induced damage by improving GSH concentrations and mitochondrial activity and cellular proliferation, inhibiting inflammation and apoptosis, and regulating PI3K and mTOR signaling pathway.

Uso da ultrassonografia Doppler colorido na reprodução de pequenos ruminantes / Use of color Doppler ultrasonography in the reproduction of small ruminants

A ultrassonografia (US) Doppler colorido fornece uma ferramenta não invasiva valiosa para detectar e monitorar mudanças dinâmicas na rede vascular e fluxo sanguíneo em vários órgãos e tecidos reprodutivos. Em apoio as biotécnicas da reprodução animal assistida, a US Doppler colorido tem mostrada alta eficiência no monitoramento funcional das estruturas ovarianas. A previsão de respostas ovarianas e produções embrionária em ovinos pela identificação de sinais Doppler na parede folicular já se mostrou eficiente. A aplicação da US Doppler colorido para a identificação da funcionalidade do tecido luteal é ainda maior, desde acompanhamentos fisiológicos e diagnósticos de disfunções luteais até ampla aplicação em conjunto as diferentes biotécnicas reprodutivas. Destaca-se em ovelhas e cabras, a aplicação comercial da US Doppler colorido para o diagnóstico de gestação precoce, de disfunções luteais, de determinação de respostas ovarianas em fêmeas doadoras e receptoras de embriões, para identificar efeitos luteotrófico de estratégias hormonais, e ainda para amparar as estratégias de ressincronização de estro.(AU)

Color Doppler ultrasonography (US) provides a valuable non-invasive tool for detecting and monitoring dynamic changes in the vascular network and blood flow in various reproductive organs and tissues. In support of assisted animal reproduction biotechniques, color Doppler US has shown high efficiency in the functional monitoring of ovarian structures. The prediction of ovarian responses and embryonic production in sheep by identifying Doppler signals in the follicular wall has already proved to be efficient. The application of color Doppler US for identifying the functionality of the luteal tissue is even greater, from physiological monitoring and diagnosis of luteal dysfunctions to wide application together with different reproductive biotechniques. It stands out in sheep and goats, the commercial application of color Doppler US for the diagnosis of early pregnancy, luteal dysfunctions, determination of ovarian responses in embryo donor and recipient females, to identify luteotrophic effects of hormonal strategies, and even to support estrus resynchronization strategies.(AU)

Dr Association of bone morphogenetic protein 15 and growth differentiation factor 9 with litter size in livestock: a review study

Litter size is one of the crucial factors in livestock production and is of high economic value, which is affected by ovulation rate, hormones, and growth factors.Growth factors play a multifaceted role in reproductive physiology. This review aims to investigate the association of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9)with litter size in livestock.The transforming growth factor ß(TGF-ß) superfamily includes more than 34 members; GDF9 and BMP15 are among the most significantfactors for regulating fertility and litter size in most livestock species. Ovarian follicles release BMP15 and GDF9 that are involved in the maturation of primary follicles into the basal form, proliferation of granulosa and theca cells, steroidogenesis, ovulation, and formation of the corpus luteum. Besides, these factors are highly expressed in oocytes and are necessary for female fertility and multiple ovulation in several livestock species. Animals with two inactive copies of these factors are sterile, while those with one inactive copy are fertile. Thus, the present reviewprovides valuable information on the association of BMP15 and GDF9with litter size in livestock that can be used as biological markers of multiple ovulation or for improving fertility in livestock.(AU)

Ovarian contrast-enhanced ultrasonography and Doppler fluxometry in bitches during the postovulatory estrus and corpora lutea formation.

The aim of the present study was to evaluate the vascularization features of canine ovaries during the follicular phase and the formation of the corpora lutea by using Doppler ultrasonography and Contrast-Enhanced Ultrasound (CEUS). Eight healthy bitches were enrolled in the study and were evaluated at five different timepoints (T1 - T5) of the estrous cycle established by vaginal cytology and serum progesterone concentration. Ultrasonographic examinations were performed by a single operator using the ACUSON S2000/SIEMENS machine equipped with a linear multifrequency transducer (9.0 MHz). Color-coded Doppler evaluation of the ovarian parenchyma was performed to investigate the aspects of the signal detection throughout the different timepoints. Pulsed-wave Doppler of the intraovarian arteries was performed to evaluate spectral waveform and doppler velocimetric parameters of Systolic Peak Velocity (SPV cm/s), End Diastolic Velocity (EDV cm/s), Resistivity Index (RI) and Pulsatility Index (PI). CEUS evaluation of the ovaries was performed using a vascular contrast agent (SonoVue®, Bracco, Sao Paulo, Brazil) and the CADENCE™ Contrast Pulse Sequencing (CPS, Siemens) software, in order to perform both qualitative and quantitative analysis. Perfusion parameters of pixel number, peak intensity (PPI in %), time to peak intensity (TTP in s), mean transit time (MTT in s) and area under the curve (AUC in %). Colour-coded Doppler evaluation demonstrated an increase in signal detection as cycle progressed, with blood flow initially detected with few coloured pixels and mainly at the ventral surface of the ovaries. Further on, the number of coloured pixels increased and spreading to the central region, resulting in a circular-like pattern around the corpora hemorrhagica. The spectral waveform was consistent at all timepoints. SPV (cm/s) and EDV (cm/s) presented a numeric trend and a slight statistical difference at all timepoints, whereas no difference was found for RI and PI. CEUS evaluation demonstrated an increase in pixel intensity across all the timepoints. Quantitative CEUS analysis revealed a statistical difference in PPI (%), MTT (s) and AUC (%) at T5. CEUS evaluation of the ovaries was feasible and demonstrated a marked increase in perfusion parameters in the late postovulatory period, demonstrating its applicability in the assessment of canine corpora lutea development.

Amburana cearensis leaf extract protects against cisplatin-induced ovarian damage through regulation of p-PTEN and p-Akt proteins in mice.

Objectives: To evaluate the effects of Amburana cearensis leaf extract against cisplatin-induced ovarian toxicity in mice and involvement of p-PTEN and p-Akt proteins. Materials and Methods: A. cearensis ethanolic leaf extract was analyzed by high-performance liquid chromatography (HPLC). Mice were pretreated once daily for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by intraperitoneal (IP) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (positive control), with (4) 50, or (5) 200 mg/kg body weight of A. cearensis extract, followed by injection of 5 mg/kg body weight (IP) of cisplatin. The ovaries were harvested and destined for histological (follicular morphology), immunohistochemistry (apoptosis and cell proliferation), and fluorescence (reactive oxygen species [ROS], glutathione concentrations [GSH], and active mitochondria) analyses. Furthermore, immunoexpression of p-PTEN and p-Akt was evaluated to elucidate a potential mechanism by which A. cearensis extract could prevent cisplatin-induced ovarian damage. Results: After HPLC analysis, protocatechuic acid was detected in the extract. The pretreatment with N-acetylcysteine or A. cearensis extract maintained the percentage of normal follicles and cell proliferation, reduced apoptosis and ROS concentrations, and increased GSH concentrations and mitochondrial activity compared with cisplatin treatment. Furthermore, pretreatment with A. cearensis extract regulated p-PTEN and p-Akt immunoexpression after cisplatin exposure. Conclusion: Pretreatment with A. cearensis extract prevented cisplatin-induced ovarian damage through its anti-oxidant actions and by modulating the expression of phosphorylated PTEN and Akt proteins.

Estrategias para mejorar los resultados reproductivos tras síndrome del folículo vacío: a propósito de un caso clínico

El síndrome del folículo vacío (SFV) es el fracaso total para recuperar los ovocitos después de la estimulación ovárica, a pesar de un desarrollo folicular aparentemente normal y una esteroidogénesis folicular adecuada. Se han descrito dos variantes de SFV: la forma genuina, que ocurre en presencia de niveles adecuados de hCGβ circulante o de LH en el momento de la aspiración de ovocitos, y la forma 'falsa', que se asocia a niveles séricos de hCG/LH por debajo de un umbral crítico. En nuestra paciente, tras un protocolo aceptado de estimulación ovárica con gonadotropina menopáusica humana y folitropina alfa y posterior maduración folicular con coriogonadotropina alfa no se obtuvieron cúmulos ovocitarios en la punción ecoguiada, con lo que se trató de emplear otras estrategias encaminadas a corregir esta situación. El tratamiento y el pronóstico de estas pacientes aún no se conocen bien. Se necesitan grandes estudios multicéntricos y revisiones sistemáticas para aumentar la comprensión del SFV y así, su manejo, diseñando mejores estrategias como tratamos de hacer con nuestra paciente con el empleo de doble descarga para maduración ovocitaria.

Empty follicle syndrome (EFS) is the complete failure to retrieve oocytes after ovarian stimulation, despite apparently normal follicular development and adequate follicular steroidogenesis. Two variants of EFS have been described: the genuine form, which occurs in the presence of adequate circulating βhCG or LH levels at the time of oocyte aspiration, and the 'false' form, which is associated with serum hCG/ LH levels below a critical threshold. In our patient, after an accepted protocol of ovarian stimulation with human menopausal gonadotropin and follitropin alfa and subsequent follicular maturation with choriogonadotropin alfa, no oocyte clusters were obtained in the ultrasound-guided puncture, so an attempt was made to use other strategies aimed at correcting this situation. The treatment and prognosis of these patients are still poorly understood. Large multicenter studies and systematic reviews are needed to increase understanding of EFS and thus its management, designing better strategies as we tried to do with our patient with the use of double discharge for oocyte maturation.

Morphology and Biochemistry of Ovulation Morfologia e bioquímica da ovulação

Abstract The process of ovulation involves multiple and iterrelated genetic, biochemical, and morphological events: cessation of the proliferation of granulosa cells, resumption of oocyte meiosis, expansion of cumulus cell-oocyte complexes, digestion of the follicle wall, and extrusion of the metaphase-II oocyte. The present narrative review examines these interrelated steps in detail. The combined or isolated roles of the folliclestimulating hormone (FSH) and luteinizing hormone (LH) are highlighted. Genes indiced by the FSH genes are relevant in the cumulus expansion, and LH-induced genes are critical for the resumption ofmeiosis and digestion of the follicle wall. A nonhuman model for follicle-wall digestion and oocyte release was provided.

Resumo O processo de ovulação envolve modificações genéticas, bioquímicas e morfológicas múltiplas e interrelacionadas: suspensão da proliferação das células da granulosa, reinício da meiose do oócito, expansão das células do complexo cumulus-oócito, digestão da parede folicular, e extrusão do oócito. Esta revisão narrativa examina em detalhes cada um desses eventos e os principais genes e proteínas envolvidos. Mais importante, a ação combinada ou isolada do hormônio folículo-estimulante (HFE) e do hormônio luteinizante (HL) é destacada. Detalha-se o papel do HFE na expansão do cumulus e do HL na digestão da parede folicular, permitindo a extrusão do oócito na superfície ovariana. Proveu-se um modelo não humano para explicar a digestão da parede folicular.

Evaluation of follicular flushing with double lumen needle in patients undergoing assisted reproductive technology treatments.

OBJECTIVE: The purpose of this study was to investigate the possible impact of follicular flushing on the number of oocytes retrieved and oocytes in metaphase II in patients with poor ovarian response (POR) compared to direct aspiration. METHODS: This prospective, comparative, randomized single center study included 208 punctures of patients with POR, submitted to assisted reproduction technology (ART) treatments. Two groups were compared; one in which double lumen needles were used (Wallace DNS1733) for follicular flushing (n=105), and one in which single lumen needles were used (Wallace ONS1733) for direct aspiration (n=103), upon the observation of ≤ 5 follicles between 15-17 mm, ≤ 4 follicles with sizes greater than 18 mm on hCG day, and ≤ 7 recovered oocytes. RESULTS: There were no differences in age (39.07±3.88 vs. 38.11±3.43); weight (61.73±17.53 vs. 65.96±15.44); AMH (0.63±0.59 vs. 0.94±0.97); stimulation days (9.57±1.87 vs. 10.29±2.82); estradiol levels (788.94±670.82 vs. 940.16±694.69); progesterone (617.29±319.76 vs. 561.18±486.78); or number of follicles with sizes ≥18 mm (1.84±0.95 vs. 2.07±1.09). Although gonadotropin totals (1678.28±798.52 vs. 2080.45±852.36; p=0.0008), number of aspirated oocytes (3.00±2.11 vs. 3.69±2.20; p=0.02), and number of metaphase II oocytes (2.20±1.64 vs. 2.99±1.88; p=0.02) were significantly different, oocyte / follicle ratio ≥15 mm (0.93 vs. 0.98) and metaphase II oocytes / follicles ≥15 mm (0.68 vs. 0.79) were similar in both groups. The failure to capture was 16% vs. 9.8%. CONCLUSIONS: Considering that there was no difference in the oocyte per follicle ratio, follicular flushing did not increase the number of oocytes recovered from poor responders.

Induction of accessory corpus luteum in goats and sheep: From physiological basis to reproductive effects / Indução de corpo lúteo acessório em caprinos e ovinos: Das bases fisiológicas aos efeitos reprodutivos

A identificação das ondas foliculares ovarianas e de seu padrão hormonal revelou que os folículos ovarianos dominantes da onda ovulatória (FDOO) crescem em ambiente hormonal com predominância crescente de estradiol, diferentemente daqueles da primeira (FDPO) e das ondas foliculares intermediárias (FDOI), que crescem sob forte impacto da progesterona (P4). O hormônio luteinizante (LH) é considerado o hormônio gonadotrófico decisivo para direcionar se um folículo dominante ovulará (↑ LH) ou não (↓ LH). Estratégias foram desenvolvidas para aumentar o LH endógeno (administração de GnRH) ou fornecer LH exógeno de origem suína (pLH) ou, ainda, gonadotrofinas semelhantes ao LH, como gonadotrofina coriônica humana (hCG). Estas medidas são capazes de disponibilizar LH para maturação final, ovulação e/ou luteinização de FDPO ou FDOI, formando corpos lúteos acessórios (CLa). Como consequência, a P4 aumenta e favorece o estabelecimento da gestação, sobretudo em condições em que a P4 for o fator limitante para a implantação e manutenção embrionária. Em ovelhas e cabras, em diferentes estudos, a hCG foi administrada de cinco a sete dias após o início do estro e revelou que o FDPO responde positivamente à administração de hCG, formando CLa e/ou promovendo a hipertrofia do CL formado originalmente, aumentando a área luteal. O incremento da P4 normalmente acompanha o aumento de área do tecido luteal. Como efeito final e mais desejável, a gestação e o nascimento de cordeiros/cabritos também aumentam. Esses conceitos serão discutidos na presente revisão sobre indução de CLa em ovinos e caprinos.

The identification of ovarian follicular waves and associated hormonal milieux has revealed that dominant follicles of the ovulatory wave (OWDF) grow in a hormonal environment where there is an increasing predominance of estradiol, unlike first-wave dominant follicles (FWDF) and intermediatewave dominant follicles (IWDF), which grow under increasing progesterone (P4) concentrations. The luteinizing hormone (LH) is considered the decisive gonadotropic hormone to direct whether a dominant follicle will (↑ LH) or will not (↓ LH) ovulate. Based on this, strategies have been developed to either increase endogenous LH (GnRH administration) or provide exogenous LH of porcine origin (pLH) or LH-like gonadotropins, such as human chorionic gonadotropin (hCG). Such strategies are able to provide LH for final maturation, ovulation, and/or luteinization of the FWDF or IWDF, forming accessory corpora lutea (aCL). As a consequence, P4 increases and favors the establishment of pregnancy, particularly when P4 is the limiting factor for the success of the conceptus implantation and maintenance. In sheep and goats, previous studies have administered hCG five to seven days after the onset of estrus and revealed that FWDF positively respond to hCG administration by either forming aCLs and/or promoting hypertrofia of the original CL which, in turn, increases its luteal tissue area. Normally, P4 synthesis increases along with the increase in luteal tissue area. As a final and most desirable outcome, pregnancy and the birth of lambs/kids also increase. These concepts will be discussed in this review, focusing on aCL induction in sheep and goats.

Evaluation and Validation of the Six Housekeeping Genes for Normalizing Mrna Expression in the Ovarian Follicles and Several Tissues in Chicken

Expression of housekeeping genes is relatively constant in different tissues and cells by RT-qPCR analysis. Housekeeping genes (HGs) are usually utilized as the reference to evaluate and compare mRNA expression abundances of target genes in different cells or tissues sampled. However, the expression stabilities of different HGs in diverse samples may appear divergence. Currently, there is no exact reference data of HGs in hen ovarian follicular tissues during egg-laying period available yet. In this study, we detected the expression of 18SrRNA, ACTB, HOXC8, GAPDH, alpha-A, and alpha-D mRNA in the varied-size ovarian follicles (1-8 mm in diameter and F5), hearts, livers, spleens, lungs, and breast muscles of the laying hens by RT-qPCR, to analyze the results via Ct value, geNorm, Normfinder, and Bestkeeper. The data showed that the expression levels of 18SrRNA, alpha-A, and alpha-D transcripts were more significantly stable than the other three genes for normalizing mRNA expression in the hen ovarian follicles examination. Moreover, alpha-D, 18SrRNA, and alpha-A were also most suitable for the expression normalization in the tissues of the heart, liver, spleen, lung and breast muscle. In contrast, 18SrRNA has the most stable mRNA expression levels in all tissues sampled, so it can serve as an excellent inner control for the evaluation of the transcription levels in chickens. It is a remarkable fact that HOXC8 as a candidate reference should be avoided. Our study establishes a set of stably expressed candidate inner references in the hen ovarian follicles and several tissues, it firstly provided an exact data for validation of the inner references in normalizing transcription levels of a target gene in chickens.(AU)

Evaluation and Validation of the Six Housekeeping Genes for Normalizing Mrna Expression in the Ovarian Follicles and Several Tissues in Chicken

Expression of housekeeping genes is relatively constant in different tissues and cells by RT-qPCR analysis. Housekeeping genes (HGs) are usually utilized as the reference to evaluate and compare mRNA expression abundances of target genes in different cells or tissues sampled. However, the expression stabilities of different HGs in diverse samples may appear divergence. Currently, there is no exact reference data of HGs in hen ovarian follicular tissues during egg-laying period available yet. In this study, we detected the expression of 18SrRNA, ACTB, HOXC8, GAPDH, alpha-A, and alpha-D mRNA in the varied-size ovarian follicles (1-8 mm in diameter and F5), hearts, livers, spleens, lungs, and breast muscles of the laying hens by RT-qPCR, to analyze the results via Ct value, geNorm, Normfinder, and Bestkeeper. The data showed that the expression levels of 18SrRNA, alpha-A, and alpha-D transcripts were more significantly stable than the other three genes for normalizing mRNA expression in the hen ovarian follicles examination. Moreover, alpha-D, 18SrRNA, and alpha-A were also most suitable for the expression normalization in the tissues of the heart, liver, spleen, lung and breast muscle. In contrast, 18SrRNA has the most stable mRNA expression levels in all tissues sampled, so it can serve as an excellent inner control for the evaluation of the transcription levels in chickens. It is a remarkable fact that HOXC8 as a candidate reference should be avoided. Our study establishes a set of stably expressed candidate inner references in the hen ovarian follicles and several tissues, it firstly provided an exact data for validation of the inner references in normalizing transcription levels of a target gene in chickens.

Sphingosine-1-phosphate (S1P) in ovarian physiology and disease.

Sphingosine-1-phoshate (S1P) is a membrane sphingolipid involved in several physiological processes, including cell proliferation, tissue growth, cell survival and migration, inflammation, vasculogenesis, and angiogenesis. Herein, we review the most critical effects of S1P on ovarian function, including its physiological and pathophysiological effects. Based on the available evidence, S1P plays an important role in ovarian physiology, participating as an essential stimulator of follicular development in both the preantral and antral phases, as well as in ovulation and corpus luteum development. Moreover, S1P may be a good cytoprotective agent against cancer treatment side-effects (chemotherapy with or without radiation therapy). In the future, this compound may be given for fertility preservation to women undergoing cancer treatment. However, further studies are required to confirm its efficacy in ovarian protection and also its safety in terms of cancer prognosis, given the biological action of the compound. Under- or over-production of S1P may be related to ovarian pathologies.

Insulin effect during a Heatsynch protocol in dairy cows / Efeito da insulina durante um protocolo Heatsynch em vacas leiteiras

The objective of this study was to evaluate the effect of exogenous insulin administration on follicular growth, estrus presentation and conception rate during a protocol of ovulation synchronization. Dairy cows were subjected to the Heatsynch protocol, with the insertion of an intravaginal device containing 1.9 g of progesterone (CIDR) and an intramuscular injection containing 2.5 mg of GnRH on day 0. On day 7, the CIDR was removed and subjects were given 12.5 mg of dinoprost. Also on day 7, Insulin Group (IG, n = 21) animals received a subcutaneous injection containing 0.25 IU / kg of recombinant human insulin and the control group (CG, n = 25) received a 0.9% NaCl injection. On day 8, an injection of 0.5 mg of estradiol cypionate was administered to all cows. Animals were inseminated 12 hours after estrus presentation or at day 10 at fixed time. Follicular development was evaluated on days 7, 9, and 10 using transrectal ultrasonography, estrus presentation was observed between days 8 and 10, and conception rate was evaluated 30 days after AI. There were no differences in growth rate, follicular diameter, estrus presentation, and conception rate. Therefore, application of a single dose of insulin does not promoted an increase in follicular size, estrus presentation and conception rate in dairy cows.(AU)

O objetivo deste estudo foi avaliar o efeito da administração exógena de insulina sobre crescimento folicular, apresentação de cio e taxa de concepção durante um protocolo de sincronização da ovulação. As vacas holandês lactantes foram submetidas ao protocolo Heatsynch, com inserção do dispositivo intravaginal contendo 1,9 g de progesterona (CIDR) no dia 0 e uma injeção intramuscular de 2,5 mg de GnRH. No dia 7, o CIDR foi removido e foi aplicado 12,5 mg de dinoprost. Ainda no dia 7, os animais do Grupo Insulina (IG, n = 21) receberam uma injeção subcutânea de 0,25 UI / kg de insulina humana recombinante e o grupo controle (CG, n = 25) recebeu uma injeção de NaCl 0,9%. No dia 8 foi aplicado 0,5 mg de cipionato de estradiol em todas as vacas. Animais foram inseminados 12 horas após a apresentação de cio ou no dia 10 em tempo fixo. O desenvolvimento folicular foi avaliado nos dias 7, 9 e 10 por ultrassonografia transretal, a apresentação de cio foi observada entre os dias 8 e 10 e a taxa de prenhez/IA foi avaliada 30 dias após a IA. Não houve diferença quanto a taxa de crescimento e diâmetro folicular, apresentação de cio e taxa de concepção. A aplicação de uma dose única de insulina não promove o incremento no tamanho folicular, apresentação de cio e taxa de prenhez/IA em vacas de leite.(AU)

Effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex.

Oxygen concentration has been shown to influence in vitro viability and growth of ovarian follicles. The present study examined the effect of oxygen tension on in vitro development of dog follicles enclosed within the ovarian cortex. Ovaries were obtained from domestic dogs (age, 8 months to 2 years), and cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 4-well culture plate containing Eagle Minimum Essential Medium (MEM). Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability and diameter immediately after collection (Control) or after 2 or 5 days of in vitro incubation. Apoptotic cells were assessed using TUNEL assay. Comparisons of follicular viability and diameter were performed using analysis of variance followed by Tukey's test (p < 0.05). Comparisons of follicle density and apoptosis among treatments were conducted using Non-parametric Kruskal-Wallis test followed by Friedman's test (p < 0.05). No difference (p > 0.05) in follicle density was observed among groups at Day 2 of in vitro culture. However, the density of follicles within cortices cultured in 20% oxygen for 5 days significantly reduced compared to the Control and those incubated in 5% concentration. The viability of cultured follicles in all treatments decreased (p < 0.05) compared to the Control after 2 days incubation, and this value further reduced (p < 0.05) in 20% oxygen group at Day 5. There were no differences in the percentages of apoptotic follicles between the two treatment groups (p > 0.05). Nevertheless, after 5 days of culture, the percentage of TUNEL-positive follicles increased significantly (p < 0.05) in cortices incubated in 20% oxygen environment. In conclusion, our findings demonstrated that 5% oxygen level was superior to 20% concentration in sustaining in vitro viability of dog follicles enclosed within the ovarian cortex.

Fibroblast growth factor 18 regulates steroidogenesis in fetal bovine ovarian tissue in vitro.

In cattle and other species, the fetal ovary is steroidogenically active before follicular development commences, and there is evidence that estradiol and progesterone inhibit follicle formation and activation. Estradiol levels decline sharply around the time of follicle formation. In the present study, we hypothesized that FGF10 and FGF18, which inhibit estradiol secretion from granulosa cells of antral follicles, also regulate fetal ovarian steroid production. Fetuses were collected at local abattoirs, and age determined by crown-rump length measurements. Real-time polymerase chain reaction assays with RNA extracted from whole ovaries revealed that the abundance of CYP19A1 messenger RNA (mRNA) decreased from 60 to 90 days of gestation, which is consistent with the decline in estradiol secretion previously observed. Immunohistochemistry revealed the presence of FGF18 in ovigerous cords in early gestation and in oocytes later in fetal age (≥150 days). The abundance of FGF18 mRNA increased after Day 90 gestation. Addition of recombinant FGF18 to fetal ovarian pieces inhibited estradiol and progesterone secretion in vitro, whereas FGF10 was without effect. Consistent with these results, FGF18 decreased levels of mRNA for CYP19A1 and CYP11A1 in ovarian pieces in vitro. These data suggest that FGF18 may be an intraovarian factor that regulates steroidogenesis in fetal ovaries.

Intrafollicular barriers and cellular interactions during ovarian follicle development.

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.

Insulin effect during a Heatsynch protocol in dairy cows / Efeito da insulina durante um protocolo Heatsynch em vacas leiteiras

ABSTRACT: The objective of this study was to evaluate the effect of exogenous insulin administration on follicular growth, estrus presentation and conception rate during a protocol of ovulation synchronization. Dairy cows were subjected to the Heatsynch protocol, with the insertion of an intravaginal device containing 1.9 g of progesterone (CIDR) and an intramuscular injection containing 2.5 mg of GnRH on day 0. On day 7, the CIDR was removed and subjects were given 12.5 mg of dinoprost. Also on day 7, Insulin Group (IG, n = 21) animals received a subcutaneous injection containing 0.25 IU / kg of recombinant human insulin and the control group (CG, n = 25) received a 0.9% NaCl injection. On day 8, an injection of 0.5 mg of estradiol cypionate was administered to all cows. Animals were inseminated 12 hours after estrus presentation or at day 10 at fixed time. Follicular development was evaluated on days 7, 9, and 10 using transrectal ultrasonography, estrus presentation was observed between days 8 and 10, and conception rate was evaluated 30 days after AI. There were no differences in growth rate, follicular diameter, estrus presentation, and conception rate. Therefore, application of a single dose of insulin does not promoted an increase in follicular size, estrus presentation and conception rate in dairy cows.

RESUMO: O objetivo deste estudo foi avaliar o efeito da administração exógena de insulina sobre crescimento folicular, apresentação de cio e taxa de concepção durante um protocolo de sincronização da ovulação. As vacas holandês lactantes foram submetidas ao protocolo Heatsynch, com inserção do dispositivo intravaginal contendo 1,9 g de progesterona (CIDR) no dia 0 e uma injeção intramuscular de 2,5 mg de GnRH. No dia 7, o CIDR foi removido e foi aplicado 12,5 mg de dinoprost. Ainda no dia 7, os animais do Grupo Insulina (IG, n = 21) receberam uma injeção subcutânea de 0,25 UI / kg de insulina humana recombinante e o grupo controle (CG, n = 25) recebeu uma injeção de NaCl 0,9%. No dia 8 foi aplicado 0,5 mg de cipionato de estradiol em todas as vacas. Animais foram inseminados 12 horas após a apresentação de cio ou no dia 10 em tempo fixo. O desenvolvimento folicular foi avaliado nos dias 7, 9 e 10 por ultrassonografia transretal, a apresentação de cio foi observada entre os dias 8 e 10 e a taxa de prenhez/IA foi avaliada 30 dias após a IA. Não houve diferença quanto a taxa de crescimento e diâmetro folicular, apresentação de cio e taxa de concepção. A aplicação de uma dose única de insulina não promove o incremento no tamanho folicular, apresentação de cio e taxa de prenhez/IA em vacas de leite.