RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

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Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

Scientific Reports ◽

10.1038/s41598-021-82051-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shauna Kehoe ◽

Katarina Jewgenow ◽

Paul R. Johnston ◽

Susan Mbedi ◽

Beate C. Braun

Keyword(s):

Gene Expression ◽

Transforming Growth Factor ◽

Ovarian Follicle ◽

Mammalian Species ◽

Expression Patterns ◽

Ovarian Follicles ◽

Domestic Cat ◽

Transcriptional Analysis ◽

Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

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Initial and Cyclic Recruitment of Ovarian Follicles*

Endocrine Reviews ◽

10.1210/edrv.21.2.0394 ◽

2000 ◽

Vol 21 (2) ◽

pp. 200-214 ◽

Cited By ~ 34

Author(s):

Elizabeth A. McGee ◽

Aaron J. W. Hsueh

Keyword(s):

Ovarian Follicle ◽

Ovarian Follicles ◽

Follicle Development ◽

Ovarian Follicle Development ◽

Branching Points ◽

Reproductive Techniques ◽

Recruitment And Selection ◽

Cyclic Recruitment

Abstract Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. The dynamics of ovarian follicle development have interested endocrinologists and developmental biologists for many years. With the advent of assisted reproductive techniques in humans, the possibility of regulating follicle development in vivo and in vitro has gained clinical relevance. In this review, we focus upon key branching points during the development of ovarian follicles as well as factors involved in determining the eventual destiny of individual follicles. We discuss inconsistencies in the literature regarding the definitions of follicle recruitment and selection and propose to name the two major steps of follicle development as initial and cyclic recruitment, respectively. Because some of these disparities have arisen due to differences in the animal systems studied, we also compare the development of the ovarian follicles of both humans and rats. We also review the status of knowledge of several puzzling clinical issues that may provide important clues toward unlocking the mechanisms of follicle development.

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Stem cell factor promotes in vitro ovarian follicle development in the domestic cat by upregulating c-kit mRNA expression and stimulating the phosphatidylinositol 3-kinase/AKT pathway

Reproduction Fertility and Development ◽

10.1071/rd16071 ◽

2017 ◽

Vol 29 (7) ◽

pp. 1356 ◽

Cited By ~ 9

Author(s):

Paweena Thuwanut ◽

Pierre Comizzoli ◽

David E. Wildt ◽

Carol L. Keefer ◽

Nucharin Songsasen

Keyword(s):

Stem Cell ◽

Mrna Expression ◽

Stem Cell Factor ◽

Ovarian Follicle ◽

Domestic Cat ◽

Follicle Development ◽

Phosphatidylinositol 3 Kinase ◽

Akt Phosphorylation ◽

Phosphatidylinositol 3

In the present study we examined the effects of stem cell factor (SCF; 50 vs 100 ng mL–1) alone or in combination with epidermal growth factor (EGF; 100 ng mL–1) on: (1) the in vitro viability and growth of cat follicles within ovarian cortices; (2) phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) phosphorylation; and (3) c-kit and FSH receptor (FSHr) mRNA expression. At 100 ng mL–1, SCF increased (P ≤ 0.05) the percentage and size of secondary follicles after 14 days of in vitro culture and sustained AKT phosphorylation after 3 days incubation. EGF suppressed this beneficial effect and reduced (P ≤ 0.05) the percentage of structurally normal follicles and FSHr expression when combined with 100 ng mL–1 SCF. Expression of c-kit mRNA was higher (P ≤ 0.05) in the presence of 100 ng mL–1 SCF compared with fresh follicles and cohorts cultured under other conditions. A c-kit inhibitor suppressed follicle growth and reduced AKT phosphorylation. Collectively, the results demonstrate that SCF promotes cat follicle development by upregulating c-kit mRNA expression and AKT phosphorylation. EGF suppresses the stimulating effect of SCF, leading to downregulation of FSHr expression.

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Anti-Müllerian Hormone Attenuates the Effects of FSH on Follicle Development in the Mouse Ovary

Endocrinology ◽

10.1210/endo.142.11.8486 ◽

2001 ◽

Vol 142 (11) ◽

pp. 4891-4899 ◽

Cited By ~ 303

Author(s):

Alexandra L. L. Durlinger ◽

Maria J. G. Gruijters ◽

Piet Kramer ◽

Bas Karels ◽

T. Rajendra Kumar ◽

...

Keyword(s):

Ovarian Follicle ◽

Inhibitory Effect ◽

Ovarian Follicles ◽

Follicle Development ◽

Wild Type ◽

Follicle Growth ◽

Primordial Follicles ◽

Ovarian Follicle Development

Abstract Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Müllerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-type, FSHβ-, AMH-, and AMH-/FSHβ-deficient females revealed that loss of FSH expression has no impact on the number of primordial and preantral follicles, but the loss of inhibitory action of AMH on the recruitment of primordial follicles in AMH-deficient mice is increased in the absence of FSH. In conclusion, these studies show that AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that AMH is one of the factors determining the sensitivity of ovarian follicles for FSH and that AMH is a dominant regulator of early follicle growth.

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Serum deprivation affects secretory activity of cultured porcine ovarian follicles and granulosa cells and their response to hormones

Veterinární Medicína ◽

10.17221/112/2009-vetmed ◽

2009 ◽

Vol 54 (No. 10) ◽

pp. 455-460

Author(s):

A.V. Sirotkin

Keyword(s):

Granulosa Cells ◽

Ovarian Follicle ◽

Secretory Activity ◽

Ovarian Follicles ◽

Serum Deprivation ◽

Ovarian Cells ◽

Igf I ◽

Granulose Cells ◽

Vitro Model

The aim of the present study is to understand the hormonal mechanisms of the effect of malnutrition on ovarian follicle functions. For this purpose, we examined the effect of malnutrition/serum deprivation, addition of metabolic hormones and gonadotropin (IGF-I, leptin and FSH) and their combination on the release of progesterone (P4), testosterone (T), estradiol (E2) and insulin-like growth factor I (IGF-I) by cultured whole ovarian follicles and on P4 and IGF-I output by cultured granulosa cells isolated from porcine ovaries. It was observed that in ovarian follicles cultured with nutrients/serum addition of IGF-I reduced release of P4, but not of T or E2. Exogenous leptin reduced output of E2, but not of P4 or T, and increased IGF-I output. No significant effect of FSH on release of steroid hormones by isolated follicles was found. Serum deprivation did not affect release of P4, but reduced output of T and E2, and promoted IGF-I release by cultured ovarian follicles. Addition of hormones failed to prevent the effect of malnutrition on the secretory activity of cultured ovarian follicles. In cultured granulose cells, all the tested hormones promoted release of both P4 and IGF-I. Food restriction/serum deprivation reduced both P4 and IGF-I output. Additions of either IGF-I, leptin and FSH prevented the inhibitory action of malnutrition on both P4 and IGF-I release. The present observations (1) confirm the involvement of the hormones IGF-I, leptin and FSH in the control of secretory activity of ovarian cells, (2) demonstrate, that both isolated ovarian granulosa cells and whole follicles cultured in the absence of serum nutrients could be an adequate in-vitro model for studying the effect of malnutrition on ovarian secretory functions, and (3) suggest, that malnutrition could affect ovarian functions through changes in the release of ovarian hormones.

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Interpenetrating fibrin–alginate matrices for in vitro ovarian follicle development

Biomaterials ◽

10.1016/j.biomaterials.2009.06.054 ◽

2009 ◽

Vol 30 (29) ◽

pp. 5476-5485 ◽

Cited By ~ 124

Author(s):

Ariella Shikanov ◽

Min Xu ◽

Teresa K. Woodruff ◽

Lonnie D. Shea

Keyword(s):

Ovarian Follicle ◽

Follicle Development ◽

Ovarian Follicle Development ◽

Alginate Matrices

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Patterns of growth, oestradiol and progesterone released by in vitro cultured mouse ovarian follicles indicate consecutive selective events during follicle development

Reproduction ◽

10.1530/jrf.0.1130287 ◽

1998 ◽

Vol 113 (2) ◽

pp. 287-297 ◽

Cited By ~ 17

Author(s):

A. Fehrenbach ◽

N. Nusse ◽

P. L. Nayudu

Keyword(s):

Ovarian Follicles ◽

Follicle Development

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In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

Biology of Reproduction ◽

10.1093/biolre/ioaa073 ◽

2020 ◽

Vol 103 (3) ◽

pp. 455-470

Author(s):

Leah E Simon ◽

T Rajendra Kumar ◽

Francesca E Duncan

Keyword(s):

Wildlife Conservation ◽

Culture Media ◽

Ovarian Follicle ◽

Three Dimensional ◽

Ovarian Follicles ◽

Culture Methods ◽

Growth And Survival ◽

Follicle Culture ◽

Culture Techniques

Abstract Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

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Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

Animal Reproduction Science ◽

10.1016/0378-4320(95)01437-3 ◽

1996 ◽

Vol 41 (1) ◽

pp. 13-26 ◽

Cited By ~ 18

Author(s):

J.G. Gong ◽

B.K. Campbell ◽

T.A. Bramley ◽

R. Webb

Keyword(s):

Growth Factor ◽

Ovarian Follicle ◽

Ovarian Follicles ◽

Follicle Development ◽

Insulin Like Growth Factor ◽

Ovarian Follicle Development ◽

Factor I ◽

Growth Factor I ◽

Bovine Somatotrophin

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Involvement of miRNAs in equine follicle development

Reproduction ◽

10.1530/rep-13-0107 ◽

2013 ◽

Vol 146 (3) ◽

pp. 273-282 ◽

Cited By ~ 43

Author(s):

S N Schauer ◽

S D Sontakke ◽

E D Watson ◽

C L Esteves ◽

F X Donadeu

Keyword(s):

Cell Survival ◽

Granulosa Cells ◽

Follicular Fluid ◽

Ovarian Follicle ◽

Follicle Development ◽

Previous Evidence ◽

Ovarian Follicle Development ◽

Fluid Levels ◽

Follicle Selection

Previous evidence fromin vitrostudies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle anovulatory wave (n=6 mares). Follicular fluid levels of progesterone and estradiol were lower (P<0.01) in S follicles than in DO follicles, whereas mean levels of IGF1 were lower (P<0.01) in S and DA follicles than in DO follicles. Relative to DO and DA follicles, S follicles had higher (P≤0.01) follicular fluid levels of miR-145 and miR-378. In Experiment 2, follicular fluid and granulosa cells were aspirated from dominant follicles before (DO) and 24 h after (L) administration of an ovulatory dose of hCG (n=5 mares/group). Relative to DO follicles, L follicles had higher follicular fluid levels of progesterone (P=0.05) and lower granulosa cell levels ofCYP19A1andLHCGR(P<0.005). Levels of miR-21, miR-132, miR-212, and miR-224 were increased (P<0.05) in L follicles; this was associated with reduced expression of the putative miRNA targets,PTEN,RASA1, andSMAD4. These novel results may indicate a physiological involvement of miR-21, miR-145, miR-224, miR-378, miR-132, and miR-212 in the regulation of cell survival, steroidogenesis, and differentiation during follicle selection and ovulation in the monovular ovary.

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