Follicular Wall Research Articles

Cyp17a effected by endocrine disruptors and its function in gonadal development of Hyriopsis cumingii

Estrogens and androgens that coexist in the aquatic environment could potentially affect shellfish, however, endocrine disrupting effects of them in shellfish are significant. As an important aquaculture shellfish in China, Hyriopsis cumingii has remarkable economic benefits. In this study, the effects of endocrine disrupting chemicals on the steroid synthase Hc-Cyp17a in the male and female gonads of the H. cumingii were assessed by exposing juvenile mussels to cultured waters containing 17β-Estradiol (E2) and 17α-Methyltestosterone (MT) for 28 days. At the same time, the E2 content in the four stages of gonadal development, the expression changes of Hc-Cyp17a in gonadal development and its localization in the mature gonad were measured to explore the relationship between genes and hormones. The results showed that both E2 and MT at 50 ng/L and 200 ng/L could affect the transcription level of Hc-Cyp17a, which was inhibited initially and promoted in post-development. E2 content was positively correlated with gonadal development stage, which was in mussel. By tracing the expression of Hc-Cyp17a, difference was found during different developmental periods. The expression level in ovary was higher than that in testis during gonadal development of 1/ 2/ 3-year-old mussels and showed an increasing trend with age. Furthermore, the expression levels in 6 tissues of mature individuals were measured and it showed that there was a significant difference between male and female in the gonads (p < 0.01). In situ hybridization, it suggested that Hc-Cyp17a was significantly signaled in the follicular wall and oocyte of female and in the follicular membrane of testis, respectively. These results could play a vital role in assessing and understanding the effects of aquatic environment on the endocrine system of H. cumingii.

Approaches for in vitro culture of granulosa cells and ovarian follicles

Abstract The in vitro culture of ovarian follicles or cumulus-oocyte complexes (COC) is used to study the factors that regulate follicular development and may have potential use in artificial reproductive technology (ART). Before ovulation, the follicle is formed by oocyte and cell populations known as granulosa cells (GCs). These cells build the internal and external mass of the follicular wall. Oocyte growth and proliferation of the surrounding cells depend on the gap junctions between the oocyte and the GCs. Maintenance of the optimal in vitro culture system allowing for preservation of follicle architecture and granulosa-oocyte interaction may be critical for success in vitro maturation of follicles. Recently many studies have focused on a culture of GCs, which have important functions related to steroidogenesis. Granulosa cells maintained in in vitro conditions exhibit stem cell properties making it important to consider in vitro culture (IVC) methods of the GC population.

Systematic Pharmacology-Based Strategy to Explore the Molecular Network Mechanism of Modified Taohong Siwu Decoction in the Treatment of Premature Ovarian Failure.

Objective To explore the molecular network mechanism of modified Taohong Siwu Decoction (MTHSWD) to interfere with premature ovarian failure based on systematic pharmacological strategy. Methods The network pharmacology strategy was used to explore the potential mechanism of MTHSWD intervention in POF, and then it was verified through animal experiments. Mouse zona pellucida 3 was used as an antigen to subcutaneously immunize BALB/c female mice to establish an immune POF model. Mice were divided into MTHSWD low-, medium-, and high-dose groups, positive control group, model group, and normal group. After 30 days of drug intervention, ovarian tissue was taken for pathological hematoxylin-eosin (HE) staining, and immunohistochemical methods were used to detect the expression of TGF-β1 and TGF-βRII and Smad2/3 protein expression in follicular wall granular cells and ovarian tissue, respectively. Results Network pharmacology studies have shown that MTHSWD may interfere with the TGF-β signaling pathway. Animal experimental research shows that, compared with the model group, the number of ovarian mature follicles in the MTHSWD groups and the positive group was significantly increased, and the number of atresia follicles decreased. Immunohistochemistry showed that, compared with the control group, the expression of TGF-β1, TGF-βRII, and Smad2/3 in the follicular wall granulosa cells and ovarian tissues of MTHSWD groups was significantly higher than that of the model group (P < 0.05). Conclusion MTHSWD may improve the ovarian function of POF mice by upregulating the protein expression of granulosa cells TGF-β1, TGF-βRII, and Smad2/3.

The utility of detecting ovulation to predict success in ovulation induction and intrauterine insemination cycles - a prospective observational study.

The success of ovulation induction-intrauterine insemination (OI-IUI) procedures may be limited by the absence of ovulation detection. The aim of this study was to evaluate the empirical use of ultrasonography and luteal phase progesterone (P4) as ovulation indicators and determine its effect on pregnancy outcome in OI-IUI cycles. This prospective observational study, which was performed in a university setting, included 107 women with unexplained infertility. Following OI, IUI was performed 36 hours after human chorionic gonadotropin (hCG). P4 was measured 72-96 hours after hCG. At the same time, the appearance of ovaries and signs indicative of ovulation, which are decreased follicle dimensions, irregularity of follicular walls, and the presence of free fluid in the Douglas pouch, were noted. In 58 patients (54.2%), ovulation was detected at the P4 level of > 10 ng/mL. Eighty-nine patients had ultrasound images suggestive of ovulation. However, only 50 of these were confirmed ovulation as indicated using P4. Implantation was observed in a total of 13 patients (12.1%). All patients were in the ovulation detected group with P4 > 10 ng/mL (AUC: 0.750; p = 0.004). P4 of > 21.5 ng/mL detected successful ovulation and was strongly associated with implantation with 77% sensitivity and 61% specificity (OR: 9.9; 95% CI: 2.4-41.2). Body mass index (BMI) > 23.9 kg/m2 was a reliable anovulation indicator as a secondary outcome (AUC: 0.696; p = 0.02). In 45.8% of the patients, ovulation did not occur even with OI treatment. The association of progesterone measurement and ovarian ultrasound scanning between 72 and 96 hours after hCG treatment can be used to detect ovulation. In doing so, we can find the optimal treatment for patients with infertility in their next cycle.

Evaluation of ImageJ software in ultrasonic image analysis: Follicular and luteal morphological characteristics of cattle

This study was conducted to compare the effectiveness of two methodologies in evaluating B- and Doppler-mode ultrasonic images: analysis using ultrasonic software and utilizing a computer with ImageJ software. To determine if ImageJ software utilization is an efficacious alternative to ultrasonic software device- analysis, there were comparisons of values when using the two methods for morphological and vascular characteristics of follicular dynamics and luteal function in 18 crossbred cattle. From day 8 of an ovarian dynamics synchronization treatment regimen period until the time of ovulation (Day 10), B-mode and power-flow ultrasonic cineloops were obtained every 12 h to assess follicular diameter, wall area, and wall blood perfusion area. On Day 14 after ovulation, US cineloops of ovaries were obtained in B mode and power flow to evaluate various morphological and vascular characteristics of the corpus luteum (CL), including luteal diameter, luteal area, and CL blood perfusion area. Cineloops were evaluated and analyzed using ultrasonic software, and in a computer with ImageJ software. To evaluate consistency in results between the two methods, there was evaluation utilizing paired t-test, Pearson correlation coefficient, Bland-Altman plot, and Linear Regression Test procedures to calculate proportion of bias between values for measurements of variables evaluated. Results indicated none of the values for variables before and after ovulation differed (P > 0.05). It, therefore, was concluded that utilization of ImageJ software is an efficacious biomedical technique to analyze ultrasonic images of morphological and vascular characteristics before and after ovulation in cattle.

Expression of kisspeptin and its receptor in different functional classes of ovarian follicle in the buffalo (Bubalus bubalis)

Recently, we reported the differential expression of kisspeptinergic system in the bubaline hypothalamus and corpus luteum. Here, we document the expression of kisspeptin (Kp) and its receptor (Kiss1r) in the ovarian follicles of the buffalo with respect to the functional status. Follicles of ≥10 to ≤13 mm diameter (n = 45) were retrospectively categorized into active (n = 18), intermediate (n = 16) and atretic (n = 11) follicles based on the concentrations of intrafollicular progesterone (P4) and estradiol (E2). The P4:E2 ratio was significantly lower in the active follicle (0.43 ± 0.08) than that of the intermediate (3.46 ± 0.53) and atretic (28.4 ± 10.6) follicles (P < 0.05). Relative fold change in the transcripts of kisspeptin (Kiss1), Kiss1r, gonadotrophin receptors, steroid acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 Family 19 subfamily A member 1 (CYP19A1), insulin like growth factor −1 (IGF-1), apoptotic factors (caspase 3 and B-cell lymphoma 2, BCL2) was calculated using qPCR in the follicular wall of the three categories of follicle (n = 8/group). In another experiment, histological sections of the ovary (n = 41) were used to group the follicles as described above and immunostaining of Kp, Kiss1r and aromatase was done. A significant upregulation of StAR, CYP11A1 and CYP19A1 in the active follicles supported the endocrine basis of follicular classification. The transcripts of Kiss1 and Kiss1r were upregulated by 19.45 fold and 4.25 fold, respectively in the active follicle as compared to other groups. Immunolocalization studies revealed that Kp and Kiss1r were localized to the basal and antral granulosa cells (GC) of the active and intermediate follicles; however, the staining intensity was stronger in the former group. Strong expression of CYP19A1 in the GC layer of active follicle supported the histological basis of defining the functional status of the follicle. It is concluded that the follicular compartment of the bubaline ovary expressed the constituents of kisspeptinergic system. The expression of Kp and Kiss1r was influenced by the functional status of the follicle with intense localization in the GC layer of the active follicles.

ANTI-FERTILITY ACTIVITY OF LEAVES AND STEM PARTS OF ARTABOTRYS ODORATISSIMUS. (ROXB.) R.BR. ANNONACEAE

The present study was done to evaluate the effects of Artabotrys odoratissimus in regulation of fertility in female albino wistar rats. Two successive solvent extracts (Ethanolic and Aqueous) of the leaves and stem of Artabotrys odoratissimus used at two dose levels 250, 500 mg/kg and 200, 400 mg/kg respectively to evaluate antifertility in female albino wistar rats by interfering the growth and release of eggs from the ovaries (ovulation). To observe the effect of various treatments on rats the rats were divided into six groups. After 21 days treatment, it was observed that the growth rate, weight gain and obesity of the rats, violent behavior of the rats like lateral attacks, biting and the muricidal behavior significant increase in Test- Iand Test- IV groups in compared with others group. The serum cholesterol and estrogen levels were increased tremendously in the Test- I, whereas estrogen levels decreased when compared to other groups. In histopathology of ovary the area of the largest follicle and the thickness of its follicular wall that is the theca cell layer and granulosa cell layer were calculated. The ovarian follicles and corpora lutea (CL) at different stages of development and regression, and the theca and granulosa cell layer were analyzed profoundly

Evaluation of developmental competence of Sus scrofa domesticus (L.) oocyte-cumulus complexes after intra- and extraovarian vitrif ication.

The aim of the present study was to identify the inf luence of extra- (EOV) and intraovarian vitrif ication (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 – 0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG); CPS-2 – 1.4 M DMSO + 1.8 M EG; CPS-3 – 2.8 M DMSO + 3.6 M EG + 0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 – 7.5 % EG + 7.5 % DMSO, then in CPS-2 – 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrif ication, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV – in 0.5 and 0.25 М trehalose. Oocytes were cultured in NCSU-23 medium with 10 % f luid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Signif icant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrif ied groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells f inished the maturation ( p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4 ± 7.5 μA and 149.2 ± 11.3 μA, respectively, p ≤ 0.05). For the f irst time, pre-implantation embryos were obtained from oocytes treated by IOV.

Expression profile of the Kisspeptin/Kiss1r system and angiogenic and immunological mediators in the ovary of cyclic and pregnant cats

The Kisspeptin/Kiss1r system has been studied in mammalian ovaries. However, there are still no studies on the modulation of this system and its relationship with angiogenic and immunological mediators in the ovary of domestic cats, especially during pregnancy. We evaluated the expression of Kisspeptin/Kiss1r and angiogenic and immunological mediators during folliculogenesis, luteogenesis and luteal regression of cyclic and pregnant cats. The ovary exhibited moderate to intense expression for Kiss1, VEGF, Flk-1, INFγ and MIF in oocytes and the follicular wall, while Kiss1r expression was low in granulosa cells. In these cells, there was also a greater expression of Kiss1, INFγ and MIF, mainly in secondary follicles, while tertiary and preovulatory follicles exhibited greater expression of VEGF and Flk-1 in this layer. In luteogenesis, Kiss1 immunostaining was higher in mature corpora lutea (MCL) of pregnant cats compared to vacuolated CL (VCL) and corpus albicans (CA). Pregnancy also increased the luteal gene expression of Kiss1 as well as Kiss1, Kiss1r, Flk-1, and MIF immunostaining in MCL, while reduced the area of VEGF expression in VCL and luteal mRNA expression of Mif when compared to non-pregnant animals. In addition, positive gene correlation between Kiss1r and Mif was observed in the CL. Kiss1, Kiss1r, Vegf and Mif expression were lower in the CA of cats in anestrus. These findings reveal that the expression of Kisspeptin/Kiss1r and angiogenic and immunological mediators, in the ovary of domestic cats, depend on the follicular and luteal stage, and the luteal expression of these mediators is influenced by pregnancy.

Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes.

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.

Response of the matrix metalloproteinase system of the chicken ovary to prolactin treatment

The expression and activity of several matrix metalloproteinases (MMPs) has been demonstrated in the chicken ovary during various physiological states; these data indicate that MMPs are involved in the remodeling of the extracellular matrix (ECM) during follicle development, ovulation, atresia, and regression. The regulation of MMPs in the avian ovary, however, remains largely unknown. The present study aimed to examine the effect of recombinant chicken prolactin (chPRL) treatment on the expression of selected MMPs and their tissue inhibitors (TIMPs), as well as MMP-2 and MMP-9 activity in the hen ovary. Real-time polymerase chain reaction revealed changes in the mRNA expression of MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, TIMP-2, and TIMP-3 in the following ovarian follicles: white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1). Western blot analysis showed alterations in the abundance of latent and active forms of the MMP-2 protein, as well as the abundance of the MMP-9 protein. Moreover, minor changes in MMP-2 and MMP-9 total activities were found in ovarian follicles of chPRL-treated hens. The response to chPRL treatment depended upon the stage of follicle development, the layer of follicular wall, and the type of MMPs or TIMPs studied. In general, the results indicate that chPRL, is a positive regulator of MMP expression in the yellow preovulatory follicles. Our findings suggest that PRL participates in the mechanisms orchestrating ECM turnover during ovarian follicular development in the hen ovary via regulating the transcription, translation, and/or activity of some constituents of the MMP system.

Decellularization Methods of Ovary in Tissue Engineering.

The ovaries or female gonads are situated in the ovarian fossa of the abdominal cavity. These are paired, almond-shaped organs measuring about 3.5cm long and 1.5cm thick and exist out of a central medullary zone and a peripheral cortex that are enclosed in a fibrous capsule called the tunica albuginea. The ovaries serve 2 main functions, the first one being the production of female gametes called oocytes (oogenesis). Interestingly, the number of primary oocytes that reside in the ovary is determined at birth. About 400 oocyte-containing follicles successfully go through all the developmental stages from this limited pool during folliculogenesis throughout the female reproductive life. In this process, primordial follicles grow and advance until forming a mature or Graafian follicle; during ovulation, secondary oocytes are released and the remaining follicular wall collapses and forms the highly vascularized corpus luteum or luteal gland. This ovarian cycle is regulated by several hormones secreted from the adenohypophysis and lasts about 28days. During this cycle, the ovaries also serve as endocrine glands and produce female sex hormones such as estrogens and progesterone (steroidogenesis), influencing the growth and development of tissues sensitive to these hormones such as the endometrium. Hence, the endometrial cycle goes synchronized with the ovarian cycle.

Ovarian gene expression in collared peccary (Pecari tajacu Linnaeus, 1758) subjected to gonadotropin stimulation protocols.

Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n=8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFβR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p>.05) between SWD (4.00±1.17) and ALD (2.50±0.43) group. The total number of follicles per animal and amounts of small (<3mm), medium (3-5mm) and large (>5mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFβR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.

Effect of eCG treatment on gene expression of selected matrix metalloproteinases (MMP-2, MMP-7, MMP-9, MMP-10, and MMP-13) and the tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3) in the chicken ovary

Several metalloproteinases (MMPs) are present and functional in the chicken ovary and regulate the extracellular matrix (ECM) during follicle development, ovulation, atresia, and regression. The regulation of the abundance of MMPs in avian ovarian follicles, however, is largely unknown. The aim of the present study was to examine effects of equine chorionic gonadotropin (eCG) on abundance of selected MMPs and relevant tissue inhibitors of MMPs (TIMPs) in the hen ovary. The MMP-2 and MMP-9 activity was also determined. Results indicated there were effects of eCG on abundances of MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, TIMP-2, and TIMP-3 mRNA transcript and/or protein relative abundances in white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1) ovarian follicles. The response to eCG depended on the stage of follicle development, layer of follicular wall, and the type of MMPs or TIMPs affected by eCG. Furthermore, there was a pause in egg laying when eCG was administered and there were morphological changes in the ovary following eCG treatment that were associated with alterations in MMP-2 and MMP-9 activity. In general, the results indicate that eCG, which has primarily follicle stimulating hormone (FSH)-like bioactivities, is a negative regulator of MMP abundance and activity in the largest yellow preovulatory follicles. Results from the present study indicate the gonadotropins, especially FSH, by the regulation of transcription, translation, and/or activity of proteins of the MMP system have effects on the mechanisms that underlie ECM remodeling and cell function throughout ovarian follicle development in the chicken ovary.

Follicular and luteal morphofunctionality of dairy cows supplemented with calcium salts of fatty acids in AIFT programs

We aimed to define the effect of strategic supplementation with calcium salts of fatty acids on the morphofunctionality of ovarian structures in crossbred dairy cows submitted to a hormonal protocol for artificial insemination at a fixed time (AIFT) A total of 20 lactating crossbred females (Bos taurus x Bos taurus indicus) were randomly divided into two groups, the control group (CON), which received concentrate with no added fat in the diet, and the treatment group (TRAT), which received concentrate with salts of fatty acid calcium (Megalac-E, Church and Dwight Co. Inc., Trenton, NJ, marketed in Brazil by Elanco-Eli Lilly and Company, Indianapolis, IN, USA). The diets were provided for 30 days. The animals started consuming the supplement 20 days before the beginning of the follicular wave and ovulation synchronization protocol (Day-20). Follicular development was monitored by mode B ultrasound and power-doppler assessments every 12 h between day D8 and the time of ovulation. By measuring the follicular diameter (DFOL), the area of the follicular wall (AFOL) and the vascularization area of the follicle wall (VFOL) were able to be determined. On days 7 and 16 after ovulation, US mode B and doppler were performed in order to analyze the corpus luteum diameter (DCL), area of the corpus luteum (ACL), and the vascularization area of the corpus luteum (VCL). In addition, blood samples were collected to measure the serum concentration of P4 using chemiluminescence methodology. There was no effect of supplementation on milk production results (p = 0.15), body weight (p = 0.57), ECC (p = 0.78), albumin (p = 0.83), and the lipid profile (CT [p = 0.36], HDL [p = 0.11], LDL [p = 0.55], VLDL [p = 0.15] and TRIG [p = 0.15]). The supplementation of SCAG, 30 days before IATF, under the conditions of this experiment had no significant influence on follicular and luteal morphofunctionality. However, suspension of supplementation caused a deficit in the luteal blood flow.

First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

Oocytes and hypoxanthine orchestrate the G2-M switch mechanism in ovarian granulosa cells.

In mammalian growing follicles, oocytes are arrested at the diplotene stage (which resembles the G2/M boundary in mitosis), while the granulosa cells (GCs) continue to proliferate during follicular development, reflecting a cell cycle asynchrony between oocytes and GCs. Hypoxanthine (Hx), a purine present in the follicular fluid, has been shown to induce oocytes meiotic arrest, although its role in GC proliferation remains ill-defined. Here, we demonstrate that Hx indiscriminately prevents G2-to-M phase transition in porcine GCs. However, oocyte-derived paracrine factors (ODPFs), particularly GDF9 and BMP15, maintain the proliferation of GCs, partly by activating the ERK1/2 signaling and enabling the G2/M transition that is suppressed by Hx. Interestingly, GCs with lower expression of GDF9/BMP15 receptors appear to be more sensitive to Hx-induced G2/M arrest and become easily detached from the follicular wall. Importantly, Hx-mediated inhibition of G2/M progression instigates GC apoptosis, which is ameliorated in the presence of GDF9 and/or BMP15. Therefore, our data indicate that the counterbalance of intrafollicular factors, particularly Hx and oocyte-derived GDF9/BMP15, fine-tunes the development of porcine follicles by regulating the cell cycle progression of GCs.

Polimorfismos del gen del receptor para la hormona luteinizante (LHR – LHGCR) en folículos ováricos de vacas Romosinuano y Brahman ciclando

El gen del receptor para la hormona luteinizante (LHR-LHCGR) es un gen altamente conservado y su mRNA origina diferentes variantes por corte y empalme alternativo (splicing alternativo). En bovinos se han reportado variantes que traducen en proteína pero ésta no atraviesa la membrana celular; en humanos ante una deleción del exón 10 no hay respuesta a la acción de la LH, por lo tanto, es razonable asumir que las diferencias en la respuesta a la LH estén relacionadas con la expresión de los LHR. El objetivo de este trabajo fue comparar entre la raza criolla Colombiana Romosinuano y la raza Brahman los posibles polimorfismos del ARNm del gen como potenciales indicadores de fertilidad. Se tomaron 5 vacas ciclando de cada raza se extirpó el ovario que contenía un folículo preovulatorio, se retiró la pared folicular, se extrajo el RNA total y se sintetizó, amplificó, secuenció y comparó el ADNc contra secuencias ya reportadas, presentando una alta similitud. Se concluye que en estas razas la ausencia de variantes en la expresión del gen, estaría asociada a buen desempeño reproductivo.

Partial inhibition of nitric oxide synthase activity stimulates the nuclear maturation progression of bovine cumulus-oocyte complex in vitro in the presence of hemisections of the follicular walls.

This study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus-oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1-10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (-HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

Changes in ovary and testis and breeding fitness of the climbing perch, Anabas testudineus (Bloch, 1792), exposed to sub‐lethal concentrations of monocrotophos

Monocrotophos (MCP) is one of the organophosphate pesticides extensively used in agriculture and animal husbandry. The present study reports the effects of sub-lethal concentrations of MCP on gonadosomatic index (GSI), gonadal histology and breeding fitness of the fish Anabas testudineus. The fish were exposed to three sub-lethal concentrations of monocrotophos (T1: 3.5 mg/L, T2: 5.3 mg/L and T3: 10.6 mg/L) for 45 days with 12 fish (males and females in equal numbers) in each tank, and one group was kept as control (C) without any treatment. GSI decreased in all treatment groups in comparison with control. Significant decrease in fertilization and hatching rate was observed at all concentrations in comparison with control on 15, 30 and 45 days of monocrotophos exposure. The decreased vitellogenesis, disruption of follicular wall and oocyte atresia were observed in all the treated females at 30 and 45 days of exposure. Rupture of seminiferous tubules was noticed in all the treated males irrespective of exposure periods. The results thus indicated that monocrotophos could deleteriously impact the gonadal structure and function in A. testudineus, which could seriously impact the reproductive success of the animal.