Localization of follicle regulatory protein in the porcine ovary.

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.

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Morphological classification of bovine ovarian follicles

Reproduction ◽

10.1530/rep-09-0177 ◽

2010 ◽

Vol 139 (2) ◽

pp. 309-318 ◽

Cited By ~ 77

Author(s):

R J Rodgers ◽

H F Irving-Rodgers

Keyword(s):

Basal Lamina ◽

Granulosa Cells ◽

Follicular Development ◽

Basal Cells ◽

Morphological Classification ◽

Primordial Follicles ◽

Antral Follicles ◽

Different Types ◽

Biological Differences

Follicle classification is an important aid to the understanding of follicular development and atresia. Some bovine primordial follicles have the classical primordial shape, but ellipsoidal shaped follicles with some cuboidal granulosa cells at the poles are far more common. Preantral follicles have one of two basal lamina phenotypes, either a single aligned layer or one with additional layers. In antral follicles <5 mm diameter, half of the healthy follicles have columnar shaped basal granulosa cells and additional layers of basal lamina, which appear as loops in cross section (‘loopy’). The remainder have aligned single-layered follicular basal laminas with rounded basal cells, and contain better quality oocytes than the loopy/columnar follicles. In sizes >5 mm, only aligned/rounded phenotypes are present. Dominant and subordinate follicles can be identified by ultrasound and/or histological examination of pairs of ovaries. Atretic follicles <5 mm are either basal atretic or antral atretic, named on the basis of the location in the membrana granulosa where cells die first. Basal atretic follicles have considerable biological differences to antral atretic follicles. In follicles >5 mm, only antral atresia is observed. The concentrations of follicular fluid steroid hormones can be used to classify atresia and distinguish some of the different types of atresia; however, this method is unlikely to identify follicles early in atresia, and hence misclassify them as healthy. Other biochemical and histological methods can be used, but since cell death is a part of normal homoeostatis, deciding when a follicle has entered atresia remains somewhat subjective.

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Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Reproduction ◽

10.1530/reprod/118.2.221 ◽

2000 ◽

pp. 221-228 ◽

Cited By ~ 7

Author(s):

HF Irving-Rodgers ◽

RJ Rodgers

Keyword(s):

Basal Lamina ◽

Granulosa Cells ◽

Light And Electron Microscopy ◽

Single Layer ◽

Antral Follicle ◽

Basal Cells ◽

Preantral Follicles ◽

Antral Follicles ◽

Cellular Processes ◽

Innermost Layer

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles </= 4 mm in diameter with columnar basal cells and with rounded basal cells. Larger follicles had only rounded basal cells. Conventional basal laminae of a single layer adjacent to the basal granulosa cells were observed in healthy follicles at the preantral and antral stages. However, at the preantral stage, the conventional types of basal lamina were enlarged or even partially laminated. A second type of basal lamina, described as 'loopy', occurred in about half the preantral follicles and in half the antral follicles </= 4 mm diameter. 'Loopy' basal laminae were not observed in larger follicles. 'Loopy' basal laminae were composed of basal laminae aligning the basal surface of basal granulosa cells, but with additional layers or loops often branching from the innermost layer. Each loop was usually < 1 microm long and had vesicles (20-30 nm) attached to the inner aspect. Basal cellular processes were also common, and vesicles could be seen budding off from these processes. In antral follicles, conventional basal laminae occurred in follicles with rounded basal granulosa cells. Other follicles with columnar cells, and atretic follicles, had the 'loopy' basal lamina phenotype. Thus, follicles have different basal laminae that relate to the morphology of the membrana granulosa.

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Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles

Reproduction ◽

10.1530/rep.0.1230097 ◽

2002 ◽

pp. 97-106 ◽

Cited By ~ 33

Author(s):

HF Irving-Rodgers ◽

ML Mussard ◽

JE Kinder ◽

RJ Rodgers

Keyword(s):

Basal Lamina ◽

Granulosa Cells ◽

Cell Shape ◽

Type Iv Collagen ◽

Basal Cells ◽

Follicle Growth ◽

Electron Microscopic ◽

Antral Follicles ◽

Type Iv ◽

Follicle Diameter

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.

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Gene expression for LH receptor, 17α-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

Reproduction ◽

10.1530/rep.1.00464 ◽

2005 ◽

Vol 129 (4) ◽

pp. 453-461 ◽

Cited By ~ 22

Author(s):

R Braw-Tal ◽

Z Roth

Keyword(s):

Gene Expression ◽

Granulosa Cells ◽

Regulatory Protein ◽

Morphological Differentiation ◽

Lh Receptor ◽

Antral Follicles ◽

Thecal Cells ◽

Key Enzyme ◽

Theca Interna ◽

Steroidogenic Acute Regulatory

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17α-hydroxylase (17αOH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n= 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated byin situhybridization. LH-R and 17αOH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17αOH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR.In summary, our study suggests that in spite of the presence of 17αOH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.

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Atresia revisited: two basic patterns of atresia of bovine antral follicles

Reproduction ◽

10.1530/rep.0.1220761 ◽

2001 ◽

pp. 761-775 ◽

Cited By ~ 66

Author(s):

HF Irving-Rodgers ◽

IL van Wezel ◽

ML Mussard ◽

JE Kinder ◽

RJ Rodgers

Keyword(s):

Basal Lamina ◽

Granulosa Cells ◽

Immunohistochemical Study ◽

Steroid Hormone ◽

Basal Layer ◽

Follicle Development ◽

Basal Cells ◽

Apoptotic Bodies ◽

Antral Follicles ◽

Follicular Fluids

Our observations of bovine follicles indicated that the original histological classifications of atresia were inaccurate. A detailed histological, ultrastructural and immunohistochemical study of antral follicles from bovine ovaries collected from an abattoir and from animals whose large follicles had been monitored by ultrasonography was conducted to investigate this further. Nidogen and CD68 were immunolocalized to observe the follicular basal lamina and macrophages, respectively. In randomly collected ovaries, approximately one quarter of all antral follicles were undergoing antral atresia, as designated in this study. Antral atresia was characterized by early destruction of the layers of the membrana granulosa closest to the antrum, whereas the most basal cells remained intact. Numerous pyknotic nuclei were observed in the most antral layers and in the antrum close to the membrana granulosa. This is the classic description of atretic follicles and was observed at all sizes of follicle development and almost universally in large follicles (> 5 mm in diameter), including dominant follicles. Basal atretic follicles, as designated in this study, were almost as prevalent as the antral atretic follicles, and were characterized by initial destruction of the most basal layer of granulosa cells, whereas the cells in the most antral layers remained associated with each other and were predominantly healthy. Pyknotic nuclei and the nuclei of dying basal cells budded into apoptotic bodies were observed rarely. The basal lamina of basal atretic follicles was often breached by macrophages, which were phagocytosing dying basal granulosa cells. The theca was characterized by an increased deposition of collagen, and the cells were orientated randomly, rather than lying parallel to the membrana granulosa as in healthy follicles. Basal atresia occurred in small (< 5 mm in diameter) follicles only. Importantly, these basal atretic follicles were originally identified incorrectly in the literature. Thus, on the basis of the results of this study and another on the expression of steroidogenic enzymes in atretic follicles, it is suggested that the standard biochemical methods for measuring steroid hormone concentrations in follicular fluids to assess atresia should be re-evaluated.

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Gene expression and protein localisation for activin-A, follistatin and activin receptors in goat ovaries

Journal of Endocrinology ◽

10.1677/joe.1.05756 ◽

2004 ◽

Vol 183 (2) ◽

pp. 405-415 ◽

Cited By ~ 28

Author(s):

J R V Silva ◽

R van den Hurk ◽

H T A van Tol ◽

B A J Roelen ◽

J R Figueiredo

Keyword(s):

Mrna Expression ◽

Granulosa Cells ◽

Activin A ◽

Surface Epithelium ◽

Corpora Lutea ◽

Rt Pcr ◽

Primordial Follicles ◽

Antral Follicles ◽

Theca Cells ◽

Activin Receptors

We studied the protein and mRNA expression of activin-A, follistatin and activin receptors in goat ovaries to find evidence of their possible role in ovarian activity, particularly in the various stages of follicle development. Ovaries of cyclic goats were collected and then either fixed in paraformaldehyde for immunohistochemical localisation of activin-A, follistatin, activin receptors IIA/B (ActR-IIA/B) and IA (ActR-IA) proteins or used to obtain samples to demonstrate mRNA expression of activin-A (βA subunit), follistatin, ActR-IIA, -IIB, -IA and -IB, using RT-PCR. For this latter goal, primordial, primary and secondary follicles were isolated mechanically, washed to remove the stromal cells and then used for RT-PCR. In addition, oocytes, cumulus, mural granulosa and theca cells from small (<3 mm) and large (3–6 mm) antral follicles, luteal cells and surface epithelium were collected to study mRNA expression. Activin-A and follistatin proteins were found in oocytes of all follicle classes, granulosa cells from the primary follicle stage onwards, theca cells of antral follicles, corpora lutea and ovarian surface epithelium. In antral follicles, these proteins were detected both in cumulus and mural granulosa cells. ActR-IIA/B protein was found at the same follicular sites, and also in granulosa cells of primordial follicles onward. The localisation of ActR-IA corresponded with that of ActR-IIA/B, but the former protein was absent in the theca of large antral follicles. The mRNAs for activin-A (βA subunit), follistatin, and ActR-IIA, -IIB, -IA and -IB were detected at all follicular and cellular types studied, except that ActR-IIB was not found in follicles that had not developed an antrum yet. In conclusion, in goat ovaries, transcripts of activin-A (βA subunit), its receptors and its binding protein follistatin are expressed and their proteins formed at all follicular stages and in corpora lutea. These findings indicate a role of activin-A in the local regulatory system during the entire follicular development and during luteal activity.

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Quantification of Ovarian Granulosa Cells and Different Level Follicles in the Mouse Ovary Using Stereology Method, for Potential Application in 3D Bioengineered Ovary.

10.21203/rs.3.rs-26397/v1 ◽

2020 ◽

Author(s):

Jiahua Zheng ◽

Jingkun Zhang ◽

Yanpeng Tian ◽

Yanbiao Song ◽

Zhengwei Yang ◽

...

Keyword(s):

Granulosa Cells ◽

Volume Fraction ◽

Three Dimensional ◽

Accurate Method ◽

Quantitative Information ◽

Numerical Density ◽

Primordial Follicles ◽

Antral Follicles ◽

Ovarian Granulosa Cells ◽

Three Stages

Abstract Background Stereology is an accurate method to obtain 3D quantitative information of microstructure based on the comprehensive observation of two-dimensional sections. Three-dimensional bioprinting of bioink to rebuild a bioengineered ovary has been proved to be a promising technique in preserving fertility. Fully understand the structure of the ovary, the distribution of the main cells and the numerical density of cells using stereology method is of great significance to the computer 3D reconstruction technology.Results The Cavalieri method gave the estimations that the ovary was (2.49 ± 0.32)mm3, the volume fraction of cortex, medulla, follicles and ovarian granulosa cells ( OGCs ) were 86.80%±2.82%, 13.20%±2.82%, 5.60%±0.25% and 81.19%±2.57%, respectively. From exact counts on serial sections, OGCs, primordial, preantral and antral follicles numerical density were estimated at ( 2.11 ± 0.28 ) × 106/mm3, 719.57 ± 18.04/mm3, 71.84 ± 3.93/mm3 and 17.29 ± 3.54/mm3, respectively. We estimated that there would be (2.11 ± 0.28) × 109 OGCs, (719.57 ± 18.04) × 103 primordial follicles, (71.84 ± 3.93) × 103 preantral follicles, (17.29 ± 3.54) × 103 antral follicles in one milliliter 3D bioink when 3D encapsulated mice ovarian constructs. The total number of the three stages follicles per ovary were 1791.74 ± 5.77, 178.88 ± 1.26, 43.04 ± 1.13, and the respective total number of OGCs and follicles per ovary were (5.26 ± 0.09) × 106, 2013.66 ± 8.16.Conclusions OGCs and follicles can be accurately measured using either the optical or physical disector. The application of these approaches for the study of 3D bioengineered ovary may contribute to a deeper understanding of the artificial ovary.

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Classification of small type B/C follicles as primordial follicles in mature rats

Reproduction ◽

10.1530/reprod/119.1.43 ◽

2000 ◽

pp. 43-48 ◽

Cited By ~ 30

Author(s):

S Meredith ◽

G Dudenhoeffer ◽

K Jackson

Keyword(s):

Dna Synthesis ◽

Granulosa Cells ◽

Single Layer ◽

Reliable Indicator ◽

Type B ◽

Primordial Follicles ◽

Type C

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.

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Immunofluorescence Assay Using Monoclonal and Polyclonal Antibodies for Detection of Staphylococcal Enterotoxins A in Milk

The Open Biotechnology Journal ◽

10.2174/187407070190130137 ◽

2019 ◽

Vol 13 (1) ◽

pp. 137-145 ◽

Cited By ~ 1

Author(s):

Tzonka Godjevargova ◽

Zlatina Becheva ◽

Yavor Ivanov ◽

Andrey Tchorbanov

Keyword(s):

Monoclonal Antibody ◽

Magnetic Nanoparticles ◽

Polyclonal Antibody ◽

Polyclonal Antibodies ◽

Food Poisoning ◽

Staphylococcal Enterotoxin ◽

Staphylococcal Enterotoxin A ◽

Fluorescence Immunoassay ◽

Magnetic Separator ◽

Milk Samples

Objectives: Staphylococcus aureus is a Gram-positive microorganism. S. aureus can grow in various foods and cause food poisoning by secreting enterotoxins. The most common enterotoxins involved in food poisoning are staphylococcal enterotoxin A and staphylococcal enterotoxin B, but Staphylococcal Enterotoxin A (SEA) is predominant. The main types of food contaminated with SEs are meat and meat products, poultry and eggs, milk and dairy products. The aim of this study was to develop a rapid and sensitive fluorescence immunoassay for detection of staphylococcal enterotoxin A in milk. Methods: Monoclonal and polyclonal antibodies for SEA were produced and characterized. Competitive fluorescence immunoassay based on Magnetic Nanoparticles (MNPs) was performed and optimized. MNPs were used as a solid carrier of the antibodies. The first step of the assay was immunoreaction between the immobilized antibody onto MNPs and SEA in milk sample. Then the fluorescein-SEA conjugate was added to the sample. Thus, competitive immunoreaction between MNP-mAb/MNP-pAb with SEA and SEA-FITC was performed. These immuno-complexes were separated by a magnetic separator and the obtained supernatants were analyzed. The fluorescent signal from the excess of conjugated SEA was proportional to the SEA contained in the milk. The assay duration was only 30 min. Results: The fluorescence immunoassays performed with polyclonal antibody had linear ranges from 5 pg/mL to 100 ng/mL SEA in a buffer, and from 50 pg/mL to 50 ng/mL SEA in spiked milk samples. While the same assays performed with monoclonal antibody had linear ranges from 1 pg/mL to 20 ng/mL SEA in buffer, and from 10 pg/mL to 10 ng/mL SEA in spiked milk samples. The detection limits of the developed immunoassays performed in milk were: 48 pg/mL with polyclonal antibody and 9 pg/mL with monoclonal antibody. Conclusion: A rapid and sensitive fluorescence immunoassay based on magnetic nanoparticles with a polyclonal and monoclonal antibody for determination of staphylococcal enterotoxin A in milk was developed.

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