Granulosa Layer Research Articles

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Antioxidant activity of self-nanoemulsifying drug delivery system (SNEDDS) of Curcuma longa in polycystic ovary syndrome rat model

Purpose: To investigate the effect of SNEDDS containing Curcuma longa extract on hormonal and histological improvement of polycystic ovary syndrome (PCOS) rat model. Method: A total of 36 female Wistar rats, aged between 8 and 10 weeks, were randomly divided into 6 groups, including control (normal rats), PCOS (untreated PCOS rats), PCOS + M (treated with 20 mg/kg bw/day metformin), PCOS + SC25, PCOS + SC50, and PCOS + SC100 (treated with Curcuma longa extract at 25, 50, and 100 mg/kg/day with SNEDDS, respectively). This study used a posttest-only control group design. The rats were euthanized on day 15, and blood samples were taken to examine malondialdehyde (MDA) and anti-mullerian hormone (AMH) levels. Ovarian tissue was prepared on a histological slide, and various follicles were observed. Data was analyzed using analysis of variance (ANOVA) and Kruskal-Wallis tests. Results: Curcuma longa extract, at 50 and 100 mg/kg, incorporated in SNEDDS resulted in a significant reduction in the corpus luteum number and width of the granulosa layer (p < 0.05). Also, Curcuma longa significantly reduced malondialdehyde (MDA), anti-mullerian hormone (AMH), preantral follicles, and follicular cysts (p < 0.05). Conclusion: Curcuma longa extract in SNEDDS reduces MDA and AMH levels and improves the histology of the ovaries in PCOS model rats. There is a need to conduct further studies of C. longa extract or isolates in SNEDDS using human cell lines of PCOS model.

Immunohistochemical Changes of Polycystic Ovaries in rats Treated with Asparagus Officinalis L. Roots Extract

Abstract Background: Polycystic ovary syndrome (PCOS) which a series reproductive, metabolic and psychological condition that is characterized by abnormal gonadotropin secretion or hyperandrogenism and associated with insulin resistance and most commonly found in the reproductive age female. Objectives: This research paper were conducted to examine the effects of Asparagus officinalis L. roots extract on letrozole induced PCOS where the effect of 400 mg/kg A. officinalis L. root extract on the PCOS was studied for 28 days in female rats. Materials and Methods: Thirty-five female Albino rats were randomly divided into five groups, including control negative group, positive control, PCOS, treatment and preventive group. During 28 days, the positive control group got an extract of the A. officinalis roots. Letrozole 1 mg/kg dissolve in 0.5% carboxymethyl cellulose solution was used to cause PCOS for 28 days. Following the induction of PCOS, the treatment group received extract A. officinalis roots for 28 days. The preventative group received an extract of asparagus roots during PCOS induction. Results: Immunohistochemical results are shown Ovary slices from of the therapeutic and preventative groups next to the asparagus group were immunostained for Ki 67, which showed high positive statistically (P <0.05) immunological activity in the granulosa nuclei and less in the theca cells. When compared to the PCOS group, both the granulosa and theca layers had very weak positive immunostaining (P <0.05). According to the morphological findings, granulosa cells from the Control group and asparagus group proliferated much more than granulosa cells from the PCOS group statistically significant (P <0.05). Conclusion: The findings show that theca and granulosa cells are quickly stimulated to proliferate by asparagus root extract. Moreover, it therefore speeds up oogenesis and early cell divisions.

MiR-21/SMAD2 Is Involved in the Decrease in Progesterone Synthesis Caused by Lipopolysaccharide Exposure in Follicular Granulosa Cells of Laying Goose.

Lipopolysaccharide (LPS) is one of the important pathogenic substances of E. coli and Salmonella, which causes injury to the reproductive system. Ovarian dysfunction due to Gram-negative bacterial infections is a major cause of reduced reproductive performance in geese. However, the specific molecular mechanisms of LPS-induced impairment of sex steroid hormone synthesis have not been determined. The regulatory mechanism of miRNA has been proposed in many physiological and pathogenic mechanisms. Therefore, the role of miRNA in breeding geese exposed to LPS during the peak laying period was investigated. In this study, twenty Yangzhou geese at peak laying period were injected with LPS for 0 h, 24 h, and 36 h. The follicular granulosa layer was taken for RNA-seq and analyzed for differentially expressed miRNAs. It was observed that LPS changed the appearance of hierarchical follicles. miRNA sequencing analysis was applied, and miR-21 and SMAD2 (SMAD family member 2) were selected from 51 differentially expressed miRNAs through bioinformatics prediction. The results showed that miR-21 down-regulated SMAD2 expression and progesterone (P4) production in LPS-treated goose granulosa cells (GCs). It also determined that overexpression of miR-21 or silence of SMAD2 suppressed the sex steroid biosynthesis pathway by decreasing STAR and CYP11A1 expression. Down-regulation of miR-21 exacerbates the LPS-induced decline in P4 synthesis and vice versa. The findings indicated that miR-21 was involved in LPS regulation of P4 synthesis in goose granulosa cells by down-regulating SMAD2. This study provides theoretical support for the prevention of LPS-induced ovarian dysfunction in geese.

Ameliorative potential of rosmarinic acid in a rat model of polycystic ovary syndrome: Targeting MCP-1 and VEGF: A histological, immunohistochemical, and biochemical study.

Polycystic ovary syndrome (PCOS) is a multidisciplinary endocrinopathy that affects women of reproductive age. It is characterized by menstrual complications, hyperandrogenism, insulin resistance, and cardiovascular issues. The current research investigated the efficacy of rosmarinic acid in letrozole-induced PCOS in adult female rats as well as the potential underlying molecular mechanisms. Forty female rats were divided into the control group, the rosmarinic acid group (50 mg/kg per orally, po) for 21 days, PCOS group; PCOS was induced by administration of letrozole (1 mg/kg po) for 21 days, and rosmarinic acid-PCOS group, received rosmarinic acid after PCOS induction. PCOS resulted in a marked elevation in both serum luteinizing hormone (LH) and testosterone levels and LH/follicle-stimulating hormone ratio with a marked reduction in serum estradiol and progesterone levels. A marked rise in tumor necrosis factor-α (TNF-α), interleukin-1β, monocyte chemotactic protein-1, and vascular endothelial growth factor (messenger RNA) in the ovarian tissue was reported. The histological analysis displayed multiple cystic follicles in the ovarian cortex with markedly thin granulosa cell layer, vacuolated granulosa and theca cell layers, and desquamated granulosa cells. Upregulation in the immune expression of TNF-α and caspase-3 was demonstrated in the ovarian cortex. Interestingly, rosmarinic acid ameliorated the biochemical and histopathological changes. In conclusion, rosmarinic acid ameliorates letrozole-induced PCOS through its anti-inflammatory and antiangiogenesis effects.

Toxic Impact of Chlorpyrifos Insecticide on the Ovary of Pigeon (Columba Livia Domestica)

Modern agriculture and vector-borne diseases are responsible for the rapid increase in pesticide usage, which is harmful to animals, particularly birds. The declining in the diversity of bird’s species is a major concern Chlorpyrifos (CPF) is the commonly used insecticide in agriculture sector. Unfortunately, CPF is still being applied across the world. Objective: To evaluate the toxic impact of chlorpyrifos on the ovaries of the pigeon in relation to the reproductive toxicity. Methods: For this purpose, N=80 healthy (Columba Livia domestica) pigeons were used. Pigeons had been acclimated for fifteen (15) days and divided into four equal groups (A-D) Oral dosages 1.3 mg, 1.6 mg, and 2.1 mg per kg body weight per day of chlorpyrifos (CPF) were administered respectively to group B, C and D for 84 consecutive days and pigeons from group (A) assigned as control. Daily observations were made on stress-related signs and behavioral changes. (H & E) staining method was applied to examine histological change in the ovary of pigeons after exposure to CPF, likewise for the control. Results: Birds from groups B-D displayed notable clinical signs such as: tremors, salivation, open-mouth breathing, ruffled feathers, lethargy and watery diarrhea and reduced food intake. The Histological examinations revealed that the ovary of pigeons from the B, C, and D showed notable variations such as follicular degeneration, vacuolation, hypertrophy, and fibrosis after exposure to CPF. The intensity was on high doses as compared to control which were with normal ovarian articulation small containing big follicles of all diameters. Oocytes, thecal layers, granulosa layers (g), stromal Tissue, and nucleus contained yolk-like secondary oocytes, mature follicles. Conclusions: In current study it was concluded that, the CPF produced drastic impact on the ovary of pigeon that may be associated with alterations in reproduction.

Alterations in aquaporin 4 expression in the chicken ovary following tamoxifen treatment

Previously, we demonstrated the follicular size- and a layer of the follicular wall-dependend expression of aquaporin 4 (AQP4) in the chicken ovary. In this study, we aimed to examine the mRNA and protein expression of aquaporin 4 (AQP4) in the chicken ovary during a pause in laying induced by a tamoxifen (TMX, oestrogen receptor modulator) treatment. Ovarian white and yellowish follicles and the granulosa and theca layers of the largest yellow preovulatory follicles were harvested from control and TMX-treated (daily until the cessation of egg-laying) hens. It was found that the TMX treatment lowered the AQP4 transcript abundance in the white follicles, but increased it in the theca layer of the F3-F1 follicles. Moreover, the TMX caused a decrease in AQP4 protein abundance in the theca layer of the F2 follicle. We propose that AQP4 plays a role in the ovarian follicle development and that there is a relationship between oestradiol action and the AQP4 gene and protein expression in chicken ovarian follicles.

Nitrophenols disrupt the expression and activity of biotransformation enzymes (CYP3A and COMT) in chicken ovarian follicles in vivo and in vitro.

Nitrophenols are environmental pollutants and xenobiotics, the main sources of which are diesel exhaust fumes and pesticides. The biotransformation processes that take place in the liver are defence mechanisms against xenobiotics, such as nitrophenols. Our previous study showed that the chicken ovary is an additional xenobiotic detoxification place and that nitrophenols disrupt steroidogenesis in chicken ovarian follicles. Therefore, the present study aimed to determine the in vivo and in vitro effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on the expression and activity of phase I (CYP3A) and phase II (COMT) biotransformation enzymes in chicken ovary. In an in vivo study, hens were treated with a vehicle or 10mg PNP or PNMC/kg b.wt. per day for 6 days. In an in vitro study, prehierarchical white and yellowish follicles, as well as the granulosa and theca layers of the three largest preovulatory follicles (F3, F2 and F1), were isolated and then incubated in a control medium or medium supplemented with PNP (10-6M) or PNMC (10-6M) for 24 or 48 h. Both in vivo and in vitro studies showed that nitrophenols exert tissue- and compound-dependent (PNP or PNMC) effects on CYP3A and COMT gene (real-time PCR) protein (Western blot) expression and their activity (colorimetric methods). The inhibitory effect of nitrophenols in vivo on the activity of biotransformation enzymes suggest that the ovary has the capacity to metabolise PNP and PNMC.

Enhancement of Melanocortin-4 Receptor (MC4R) and Constancy of Kiss1 mRNAs Expression in the Hypothalamic Arcuate Nucleus in a Model of Polycystic Ovary Syndrome Rat

Background: Hypothalamic Melanocortin-4 Receptor (MC4R) and kiss1/kisspeptin systems play roles in reproductive processes. This study was conducted to evaluate changes in MC4R and kiss1 genes expression in the arcuate nucleus (ARC) of the hypothalamus and its relationship with polycystic ovary syndrome (PCOS) in rats. Materials and Methods: In the current experimental study, 24 female rats were randomly and equally allocated into nulliparous and primiparous groups and then were divided into two subgroups of PCOS and control. PCOS was induced by exposure to continuous light. Sex-related hormones were evaluated by radioimmunoassay or immunoradiometric assay. Expressions of MC4R and kiss1 gene in the ARC of the hypothalamus of the rats were evaluated by real-time PCR. Histomorphometric alterations of ovaries were compared between groups. Results: Number of tertiary follicles and their size and number of atretic follicles in the PCOS subgroups were more than those in the controls (P<0.05) whereas the number of secondary follicles and corpus luteum in the PCOS subgroups were lower than those in the controls (P<0.05). Antrum and total diameters of tertiary follicles in the PCOS subgroups were greater and granulosa layer diameter was lower than those in the controls (P<0.05). The MC4R mRNA expression in the PCOS subgroups was 6.5-fold in nulliparous and 3.5-fold in primiparous groups more than their controls’ pairs (P<0.05). However, parity did not affect the expression of MC4R gene (P>0.05).The kiss1 mRNA expression in the PCOS and control subgroups was not significantly different (P>0.05). Conclusion: Overexpression of MC4R gene after PCOS induction in the ARC of the hypothalamus may link to metabolic disorders of induced PCOS in the rats. However, alteration in the kiss1 mRNA expression after PCOS induction was not observed in the rats. [GMJ.2018;7:e1070]

Transcriptome Analysis of Granulosa Cells Reveals Regulatory Mechanisms Related to Chicken Follicle Development.

In this study, we aimed to better understand the difference between the functions of the two types of granulosa cells and sought to discover more key genes involved in follicle development and follicle selection. Herein, we separately collected pre-hierarchical follicle granulosa cells (PHGCs) and preovulatory follicle granulosa cells (POGCs) for RNA extraction; the transcriptomes of the two groups were compared via RNA-seq. A total of 5273 differentially expressed genes (DEGs) were identified between the PHGCs and POGCs; 2797 genes were up-regulated and 2476 were down-regulated in the PHGCs compared with the POGCs. A qPCR analysis confirmed that the expression patterns of 16 randomly selected DEGs were highly consistent with the RNA-seq results. In the POGCs, many of the genes with the most significant increase in expression were related to steroid hormone synthesis. In addition, the genes with the most significant decline in expression, including AMH and WT1, were related to the inhibition of steroid hormone synthesis. These results suggest that steroid hormones play a key role in follicle development. Furthermore, a Gene Ontology (GO) analysis revealed that these DEGs were mainly involved in the primary metabolic process, the carbohydrate metabolic process, the cellular process, ribosomes, the cytoplasm, and intracellular processes. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were mainly enriched in steroid biosynthesis, the cell cycle, ribosomes, the TGF-beta signaling pathway, focal adhesion, and so on. We also observed the morphology of the follicles at different developmental stages, and the results showed that the thickness of the granular layer of the small yellow follicles (SYFs) decreased significantly with further development. In addition, we also found that the thickness of the granulosa layer of hens over 300 days old was significantly lower than that of 200-day-old hens. In short, these data indicate that the tissue morphology and function of granulosa cells change throughout follicle development.

Vitamin D3 Receptors and Metabolic Enzymes in Hen Reproductive Tissues

In recent years, vitamin D3 has been revealed as an important regulator of reproductive processes in humans and livestock; however, its role in the female reproductive system of poultry is poorly known. The aim of this study was to examine vitamin D3 receptor (VDR and PDIA3) and metabolic enzyme (1α-hydroxylase and 24-hydroxylase) mRNA transcript and protein abundances, and protein localization within the hen ovary, oviductal shell gland, pituitary, liver, and kidney. We demonstrated, for the first time, the patterns of the relative mRNA and protein abundances of examined molecules in the ovary, dependent on follicle development and the layer of follicle wall, as well as in other examined organs. Immunohistochemically, PDIA3, 1α-hydroxylase, and 24-hydroxylase are localized in follicular theca and granulosa layers, luminal epithelium and tubular glands of the shell gland, pituitary, liver, and kidney. These results indicate that reproductive tissues have both receptors, VDR, primarily involved in genomic action, and PDIA3, probably participating in the rapid, non-genomic effect of vitamin D3. The finding of 1α-hydroxylase and 24-hydroxylase expression indicates that the reproductive system of chickens has the potential for vitamin D3 synthesis and inactivation, and may suggest that locally produced vitamin D3 can be considered as a significant factor in the orchestration of ovarian and shell gland function in hens. These results provide a new insight into the potential mechanisms of vitamin D3 action and metabolism in the chicken ovary and oviduct.

Comparative analyses of laying performance and follicular development characteristics between fat and lean broiler lines

The deposition of high levels of fat in broiler breeder hens can have a profound impact on follicular development and laying performance. This study was formulated with the goal of comparing egg production and follicular development characteristics at different laying stages in the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). The egg production was analyzed using the birds from the 19th to 24th generations of NEAUHLF; the follicular development characteristics were analyzed by hematoxylin-eosin staining and quantitative real-time polymerase chain reaction using the birds from the 24th generation of NEAUHLF. The results showed that the age at first egg of lean hens was significantly earlier than that of fat hens in this study. While no significant differences in total egg output from the first egg to 50 wk of age were noted when comparing these 2 chicken lines, lean hens laid more eggs from the first egg to 35 wk of age relative to fat hens, whereas fat hens laid more eggs from wk 36 to 42 and 43 to 50 relative to their lean counterparts. No differences in ovarian morphology and small yellow follicle (SYF) histological characteristics were noted when comparing these 2 chicken lines at 27 wk of age. At 35 and 52 wk of age, however, lean hens exhibited significantly lower ovarian weight, ovarian proportion values, numbers of hierarchical follicles, hierarchical follicle weight, and SYF granulosa layer thickness as compared to fat hens, together with a significant increase in the number of prehierarchical follicles relative to those in fat hens. Gene expression analyses suggested that follicle selection was impaired in the fat hens in the early laying stage, whereas both follicle selection and maturation were impaired in the lean hens in the middle and late laying stages. Overall, these data highlight that fat deposition in broiler hens can have a range of effects on follicular development and egg production that are laying stage-dependent.

Biomolecular composition of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination

The study aimed to analyze changes in biomolecular composition of granulosa and theca interna cells of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination. Medium antral follicles (n = 4/each group) were cultured alone (C; control) or in the presence of 1α,25(OH)2D3 (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination, VD and I (VD+I). Then paraplast-embedded ovarian follicles were used for Fourier Transform Infrared (FTIR) spectroscopy and respective histological stainings. FTIR analysis revealed changes in the content of fibrous proteins (mainly collagens) within theca interna following vitamin D3 and insulin co-administration that was verified by Masson’s trichrome staining. Treatment-dependent differences were also observed in the secondary structure of proteins, indicating enhanced conversion to α-helices in response to vitamin D3 and insulin action/interaction in both follicular compartments. In the granulosa and theca interna layers, tendency to lower DNA content in the VD+I group was noted and confirmed by Fulgen’s staining. Finally, altered monosaccharides production in both follicular layers was found. Based on FTIR results, it is possible to attribute the observed alterations to biological processes that could be regulated by vitamin D3 and insulin in the porcine ovarian follicles.

Luteinizing hormone stimulates ingression of mural granulosa cells within the mouse preovulatory follicle†.

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 min of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10h after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4h after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells1

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In the present study, ovarian tissues from 30 w and 15 w laying chickens were analyzed using RNA-seq to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT–PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6-8 mm) and preovulatory follicles (F1). In vitro, Follicle stimulating hormone or Luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

1α,25-Dihydroxyvitamin D3 Improves Follicular Development and Steroid Hormone Biosynthesis by Regulating Vitamin D Receptor in the Layers Model

1α,25-Dihydroxyvitamin D3 (VitD3) is the active form of vitamin D, and it regulates gene expression and protein synthesis in mammalian follicle development. However, the function of VitD3 in the follicular development of layers remains unclear. This study investigated, through in vivo and in vitro experiments, the effects of VitD3 on follicle development and steroid hormone biosynthesis in young layers. In vivo, ninety 18-week-old Hy-Line Brown laying hens were randomly divided into three groups for different treatments of VitD3 (0, 10, and 100 μg/kg). VitD3 supplementation promoted follicle development, increasing the number of small yellow follicles (SYFs) and large yellow follicles (LYFs) and the thickness of the granulosa layer (GL) of SYFs. Transcriptome analysis revealed that VitD3 supplementation altered gene expression in the ovarian steroidogenesis, cholesterol metabolism, and glycerolipid metabolism signaling pathways. Steroid hormone-targeted metabolomics profiling identified 20 steroid hormones altered by VitD3 treatment, with 5 being significantly different among the groups. In vitro, it was found that VitD3 increased cell proliferation, promoted cell-cycle progression, regulated the expression of cell-cycle-related genes, and inhibited the apoptosis of granulosa cells from pre-hierarchical follicles (phGCs) and theca cells from prehierarchical follicles (phTCs). In addition, the steroid hormone biosynthesis-related genes, estradiol (E2) and progesterone (P4) concentrations, and vitamin D receptor (VDR) expression level was significantly altered by VitD3. Our findings identified that VitD3 altered the gene expression related to steroid metabolism and the production of testosterone, estradiol, and progesterone in the pre-hierarchical follicles (PHFs), resulting in positive effects on poultry follicular development.

OXIDATIVE PROCESSES IN GRANULOSA CELLS OF COW OVARIAN FOLLICLES AT FOLLICULAR GROWTH

Article presents the research results on the respiratory activity and redox capacity of granulose layer cells of cow ovarian follicles under the physiological state of follicular growth. After the slaughter of the cows, the ovaries in physiological state of follicular growth were selected and the size of the follicles was evaluated. Cells of the granulosa layer were aspirated from small (< 4 mm), medium (4–7 mm) and large (> 7 mm) follicles and determined: intensity of oxygen absorption – polarographically, redox capacity – potentiometrically. K3[Fe(CN)6] was used as an extracellular electron acceptor. The share of oxygen consumed by aerobic glycolysis was determined by using sodium fluoride inhibitor, the NAD-dependent link of the mitochondrial electron transport chain - amytal, and the terminal (cytochrome oxidase) – sodium azide. The share of free-radical oxidation of unsaturated fatty acids in the realization of oxygen was detected by Na2EDTA.
 Granulosa cells from the ovary of follicular growth are characterized by respiratory activity and regenerative capacity, the intensity of which depends on the size of the follicles from which they are extracted. The highest values of the investigated indicators are characteristic of cells extracted from large follicles, lower – from small follicles, and the lowest values are from medium follicles. The high intensity of oxidative processes in granulosa from small follicles ensures the growth of oocytes, and from large follicles – maturation of the germ cell in the dominant follicle. At the same time, the increased intensity of oxygen consumption by the cells of small and dominant follicles is manifested against the background of limitations in electron acceptors in the extracellular environment.
 The use of inhibitors of ATP resynthesis of both glycolysis and individual links of the mitochondrial respiratory chain testify the activity of the indicated metabolic pathways in granulosa and the possibility of using alternative substrates to ensure the existence of cells. The main way of utilization of oxygen in the granulosa layer cells of small and large follicles is aerobic glycolysis, and from the medium there are alternative ways of supplying and using substrates. The largest flow of reducing equivalents into the extracellular space from the granulosa of a medium-sized follicle due to inhibition of glycolysis indicates that the source of the supply of reducing equivalents into the follicle cavity is glycolysis and this characterizes the dependence of the existence of cells on the indicated metabolic pathway.

Histomorphological Observations in Reproductive Organs of Wistar Rats Administered with Aqueous Leaf Extract of Momordica charantia

Effort to explore the adverse effect of the plant, Momordica charantia on mammals has remained inadequate despite previous attempt by earlier investigators. More glaring is the paucity of information on the histomorphological effect of the plant’s aqueous leaf extract hence the need to determine possible alteration of tissue structures in reproductive organs of adult Wistar rats which may affect their functions.
 This study aimed at determining the effect of Momordica charantia aqueous leaf extract (MCALE) on the reproductive organs of experimental adult Wistar rats.
 Materials and Methods: Twenty five rats (male and female) weighing 180-200g were randomly divided into five (5) groups of five rats each. The experimental groups, A to D were fed on standard diet and administered with 100, 200, 400 and 800 mg/kg body weight /day of MCE orally using gavage for 30 days. Rats in the control groups were fed on standard diet and physiological saline orally. Organs were harvested, fixed in 10% neutral buffered formalin (ovaries) and Bouin’s fluid (testes), embedded in molten paraffin wax, sectioned with a rotary microtome and stained with the haematoxylin and eosin technique. Stained slides were examined using the Olympus microscope. 
 Results: Sections of ovaries administered 100 mg/kg of the extract showed vesicular spaces in corpus luteum and enlarged blood vessels. Sections treated with 200 mg/kg revealed follicular cyst and mild vacuolation of zona granulosa. Sections of the ovaries administered 400 mg/kg revealed degenerative changes, follicular cyst, mild vacuolation and reduction of zona granulosa layer while those treated with 800 mg/kg showed severe vacuolation of the zona granulosa layer. 
 Conclusion: Momordica charantia caused histomorphological changes in ovaries of Wistar rats which could cause hormonal imbalance and infertility in females. No histomorphological changes were observed in male testes.

Molecular characterisation and expression profile of the PRLR gene during goose ovarian follicle development

ABSTRACT 1. Although PRL-PRLR signalling plays important roles in regulating avian reproduction, there is a paucity of information regarding the functional significance of PRLR in goose ovarian follicle development. 2. The full-length 2,496 bp coding sequence of PRLR was obtained from Sichuan White goose (Anser cygnoides) for the first time and was seen to encode a polypeptide containing 831 amino acids. Goose PRLR shares similar sequence characteristics and conserved functional domains to other avian species and was phylogenetically clustered into the avian clade. 3. The qPCR results suggested that the mRNA levels of PRLR significantly increased in primary follicles during weeks 3 to 4 of age and were higher in secondary- than in primordial follicles at week 5 post-hatching, which suggested that the PRLR-mediated signalling could be involved in regulation of early folliculogenesis. 4. The PRLR mRNA was expressed at the highest levels in the prehierarchical 8–10 mm granulosa layers throughout goose ovarian follicle development, indicating a role for PRLR in the process of follicle selection. 5. PRLR mRNA was differentially expressed in the three cohorts of in vitro cultured granulosa cells harvested from different sized goose ovarian follicles, which suggested that PRLR was involved in regulating granulosa cell functions depending on the stage of follicle development. These data provide novel insights into the role of PRLR during goose ovarian follicle development, although the underlying mechanisms await further investigations.

Global gene expression analysis of the turkey hen hypothalamo-pituitary-gonadal axis during the preovulatory hormonal surge

The preovulatory hormonal surge (PS) consists of elevated circulating luteinizing hormone (LH) and progesterone levels and serves as the primary trigger for ovarian follicle ovulation. Increased LH and progesterone, produced by the pituitary and the granulosa layer of the largest ovarian follicle (F1), respectively, result from hypothalamic stimulation and steroid hormone feedback on the hypothalamo-pituitary-gonadal (HPG) axis. The hypothalamus, pituitary, F1 granulosa, and granulosa layer of the fifth largest follicle (F5) were isolated from converter turkey hens outside and during the PS and subjected to RNA sequencing (n=6 per tissue). Differentially expressed genes were subjected to functional annotation using DAVID and IPA. A total of 12, 250, 1235, and 1938 DEGs were identified in the hypothalamus, pituitary, F1 granulosa, and F5 granulosa respectively (q<0.05, |fold change|>1.5, FPKM>1). Gene Ontology (GO) analysis revealed key roles for metabolic processes, steroid hormone feedback, and hypoxia induced gene expression changes. Upstream analysis identified a total of 4, 42, 126, and 393 potential regulators of downstream gene expression in the hypothalamus, pituitary, F1G, and F5G respectively, with a total of 63 potential regulators exhibiting differential expression between samples collected outside and during the PS (|z-score|>2). The results from this study serve to increase the current knowledge base surrounding the regulation of the PS in turkey hens. Through GO analysis, downstream processes and functions associated with the PS were linked to identified DEGs, and through upstream analysis, potential regulators of DEGs were identified for further analysis. Linking upstream regulators to the downstream PS and ovulation events could allow for genetic selection or manipulation of ovulation frequencies in turkey hens.