333. HETEROGENEITY OF GENE EXPRESSION IN BOVINE SMALL FOLLICLES

2010 ◽  
Vol 22 (9) ◽  
pp. 133
Author(s):  
N. Hatzirodos ◽  
H. F. Irving-Rodgers ◽  
R. J. Rodgers

Small antral follicles <5 mm in bovine ovaries undergo one of two fates: further growth and selection to become the dominant follicle for ovulation, or atresia. Atresia can occur before, during or after selection. As follicle grow past >5 mm there is upregulation in expression of focimatrix genes and later upregulation of the LH receptor and steroidogenic enzymes, especially aromatase, in the granulosa cells. For follicles at sizes >5 mm entering atresia the granulosa cells are the first in the follicle to die. Thus expression of genes in granulosa cells is critical to the fate of the follicle. To examine granulosa cells of small follicles we collected bovine ovaries and dissected follicles, removed part of the follicle wall for subsequent classification of health or atresia, and harvested the remaining granulosa cells for RNA isolation. Follicles examined included small follicles (<5 mm), both healthy (n = 10) and atretic (n =5), and healthy large follicles (>10 mm, n = 4). RNA was hybridized to Affymetrix GeneChip Bovine Genome Arrays and the results were analysed using Partek Genomics Suite software. The number of genes which were 2 fold differentially regulated between large and small follicles by Benjamini Hochberg post hoc test (False Discovery Rate, P < 0.05) was 2408 and between healthy and atretic small follicles was 4931. The coefficient of variation (CV; SD/mean × 100) for the expression level of each gene for each group was calculated. A gene frequency distribution indicated greater heterogeneity in expression levels in small follicles in comparison to large follicles. Furthermore, the greatest variability in genes in small follicles includes those that are either up or down regulated due to atresia or growth. We therefore conclude that variability in small follicles is a consequence of alternative fates that small follicle can undergo.

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T Okubo ◽  
H Teruaki ◽  
O Noriyuki ◽  
O Kenji ◽  
S Tomoya

Abstract Study question Do different follicle sizes influence gonadotropins (LH, FSH) and sex steroid (estradiol) in follicular fluids and LH receptor expression (LHCGR) in cumulus oocyte complexes (COCs)? Summary answer It was found that differences in levels of FSH, estradiol values and LHCGR mRNA expression level in COCs between small and large follicles. What is known already The maturity rate in oocytes of small follicle is significantly lower compared to that of large follicles. Study design, size, duration After obtaining written consents from 78 infertile patients, we aspirated the large (&gt;15 mm) and small (&lt;5 mm) follicles, and collected follicular fluids at oocyte retrieval. Participants/materials, setting, methods We measured levels of LH, FSH and estradiol by enzyme immunoassay from large and small follicular fluids after oocytes retrievals. All collected oocytes were distinguished from large and small follicles, we confirmed the maturity of retrieved oocytes by the presence of first polar body. Then we extracted total RNA from granulosa cells and measured mRNA expression of LHCGR, encoding the human LH receptor, by quantitative real-time PCR. Each value was normalized to ACTB mRNA levels. Main results and the role of chance LH levels were nearly equal between small and large follicles (P = 0.8356). Whereas FSH and estradiol levels were significantly lower in small follicles (P &lt; 0.0001). The expression levels of LHCGR mRNA were significantly lower in small follicles than in large follicles during natural cycles. The maturity rate in oocytes of small follicle was significantly lower compared to that of large follicles (96.0% vs. 21.7%, P &lt; 0001). Limitations, reasons for caution The main limitation of the present study was collected by 42 natural cycles and 36 mild stimulation cycles with letrozole following low-dose clomiphene. Wider implications of the findings: In spite of almost the same LH levels between two groups, the reason why the significantly lower maturation rates of oocytes collected from small follicles is poor LHCGR mRNA expression due to insufficient granulosa cells glowth because of low FSH and estradiol levels. Trial registration number Not applicable


Reproduction ◽  
2000 ◽  
pp. 43-48 ◽  
Author(s):  
S Meredith ◽  
G Dudenhoeffer ◽  
K Jackson

In the present study, follicles were classified according to the morphology of their granulosa cells. Type B follicles contained only flattened granulosa cells; type B/C follicles had a mixture of flattened and cuboidal granulosa cells in a single layer, and type C follicles had a single layer of cuboidal granulosa cells. The primary objectives of the study were to determine whether 5-bromo-2-deoxyuridine incorporation into type B/C follicles was a marker for initiation of growth and how long type B/C follicles could remain at the same stage before transformation to type C follicles. Female Holtzman rats received bromo-deoxyuridine for 7 days. After the infusion (day minipumps were removed = day 0), rats were ovariectomized on days 0 (n = 9), 30 (n = 8), 90 (n = 8) and 150 (n = 9). The numbers of type B, B/C and C follicles within one ovary were determined using modified fractionator counting. Analysis over all times demonstrated that there were more (P < 0.0001) type B/C (941 +/- 61 per ovary) than type C (140 +/- 18 per ovary) or type B (159 +/- 19 per ovary) follicles. The numbers of type B and type C follicles did not differ from each other at any time. Only one of 34 rats evaluated had bromo-deoxyuridine-labelled type B follicles. On day 150, 57% of the bromo-deoxyuridine-labelled type B/C follicles remained from day 0. It is concluded that (1) DNA synthesis in granulosa cells of type B/C follicles is not a reliable indicator of impending growth; and (2) type B and type B/C follicles are both components of the pool of primordial follicles.


Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.


Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.


Endocrinology ◽  
2015 ◽  
Vol 156 (9) ◽  
pp. 3192-3202 ◽  
Author(s):  
Kohshiro Nakao ◽  
Hiroshi Kishi ◽  
Fumiharu Imai ◽  
Hiroto Suwa ◽  
Takashi Hirakawa ◽  
...  

Several inflammatory cytokines regulate ovarian function. TNF-α is produced in granulosa cells under physiological conditions and has a reciprocal action on follicle development. In contrast, in pelvic inflammatory diseases, TNF-α is excessively produced in the pelvic cavity and has an adverse effect on reproductive functions. The objective of this study was to elucidate the mechanism of action of TNF-α on the expression of LH receptor (LHR) in immature rat granulosa cells. TNF-α suppressed FSH-induced LHR mRNA and protein expression and was not associated with cAMP accumulation. By using a luciferase assay, the construct containing base pairs −1389 to −1 of the rat Lhcgr promoter revealed that TNF-α decreased FSH-induced promoter activity. In response to TNF-α, nuclear factor (NF)-κB p65 was translocated to the nucleus, and the suppressive effect of TNF-α on LHR mRNA expression was abrogated by an NF-κB inhibitor. In a chromatin immunoprecipitation assay, TNF-α induced the association of NF-κB p65 with the rat Lhcgr transcriptional promoter region. NF-κB p65 and histone deacetylase (HDAC) interact to mediate expression of several genes at a transcriptional level. HDAC activity is thought to induce tight connections within local chromatin structures and repress gene transcription. Furthermore, the TNF-α–induced suppression of LHR mRNA expression was blocked by an HDAC inhibitor. Taken together, these results suggest that the interaction of NF-κB p65 with HDAC in the promoter region of rat Lhcgr might be responsible for TNF-α action on the regulation of LHR.


Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 120
Author(s):  
Rihong Guo ◽  
Fang Chen ◽  
Zhendan Shi

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.


1992 ◽  
Vol 9 (3) ◽  
pp. 309-312 ◽  
Author(s):  
P.F. Whitelaw ◽  
C.D. Smyth ◽  
C.M. Howles ◽  
S.G. Hillier

ABSTRACT Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.


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