Academic literature on the topic 'Early antral follicle'

Abstract During early pregnancy offspring are directly exposed to nutrients consumed by their mother, and the development of their own reproductive tract is underway. The objective of this research was to determine how characteristics of offspring ovaries were affected by different maternal rates of gain during the first trimester of gestation in beef heifers. Before breeding antral follicle counts were determined via ultrasound. Beginning at breeding Angus heifers were managed to achieve one of two rates of gain: low (0.20 kg/d, n = 8; LG) or moderate (0.75 kg/d, n = 8; MG) for the first trimester of pregnancy, after which they were managed as a single group through F1 calving. Calves remained with their dams until weaning and were managed as a single group through breeding. These F1 heifer calves were then synchronized and bred to a single sire using female sexed semen. On the 84th day of gestation (approximate heifer age 17 months) ovaries were removed from the reproductive tract, weighed, and visible antral follicles were counted. Cross-sections from each ovary were post-fixed and embedded in paraffin, then each of 3 sections (5 µm, with 10 sections between to avoid counting follicles more than once) were stained with Hematoxylin and Eosin for histological evaluation. Number of primordial, primary, secondary, and antral follicles were determined for each section. Data were analyzed using the GLM procedure of SAS with significance declared at P ≤ 0.05. The CORR procedure of SAS was used to calculate correlations between pre-breeding antral follicle counts, surface follicle counts, and histological follicle counts. Though no differences were observed in number of visible follicles (P = 0.45), the corpus luteum (CL) was heavier (P = 0.03) and average ovarian length was greater (P = 0.04) in offspring from LG dams compared with those from MG dams. No differences were observed in the number of primordial, primary, secondary, or antral follicles between treatments (P ≤ 0.18). There was a correlation between the number of histological follicles and surface follicles (r = 0.73; P = 0.002) and pre-breeding antral follicles and surface follicles (r = 0.60; P = 0.014), but there was no correlation between pre-breeding antral follicles and histological follicles (P = 0.10). Heavier CLs have a positive correlation with amount of progesterone released, which is essential for pregnancy maintenance. Longer lengths of ovaries suggest more area for follicles to develop, which is indicative of future reproductive success. Data corroborates previous reports regarding positive correlations between antral follicle count determined via ultrasound and counts of surface follicles on extracted ovaries. These findings demonstrate that nutrition during the first trimester of gestation impacts offspring reproductive tract development.

Abstract Several clinical studies have shown a correlation of hypersecretion of LH and polycystic ovary syndrome (PCOS), infertility, and miscarriage in women, suggesting that chronically elevated LH impairs fertility. Growth arrest of small antral follicles in PCOS is also assumed to be associated with an abnormal endocrine environment involving increased LH stimulation, a hyperandrogenic milieu, and subsequent dysregulated FSH action in the ovarian follicles. In this study, we examined whether and how LH modulates follicular development and steroid production during preantral-early antral follicle transition by using a rat preantral follicle culture system. LH augments testosterone and estradiol production in preantral follicles via up-regulating mRNA abundance of CYP17A1 and CYP19A1. LH promotes rat preantral follicle growth, and the follicular size reaches that of early antral follicles in vitro, a response attenuated by the specific androgen receptor antagonist and a targeted disruption of androgen receptor gene. Sustained follicle stimulation by LH, but not by androgen, decreases FSH receptor mRNA levels and FSH receptor signaling and inhibits FSH-induced follicular growth. The data suggest that LH promotes preantral-early antral transition via the increased synthesis and growth-promoting action of androgen. However, chronic LH stimulation impairs FSH-dependent antral follicle growth by suppressing granulosa cell FSHR expression via the modulation of intraovarian regulators, including LH-induced thecal factors.

Abstract Both estrogen receptor (ER) α and β are expressed within the ovary and lack of either of these receptors affects ovarian function. In this study, the role of ERα and ERβ in folliculogenesis and ovulation was further analyzed. Evaluation of ovarian follicle populations in wild-type and ERβ knockout (βERKO) ovaries revealed reduced late antral growth and ovulatory capacity of βERKO follicles, indicated by reduced numbers of large antral follicles and corpora lutea and increased atresia of large antral follicles. An in vitro culture system was used to study growth, rupture, and luteinization of wild-type, ERα knockout (αERKO) and βERKO ovarian follicles. αERKO follicles exhibited wild-type-like growth and ovulation rates but an increased capacity to synthesize estradiol. In contrast, βERKO follicles showed a significant lack of progression from early antral to large antral stage, decreased estradiol production, and reduced ovulation. Expression patterns of several genes involved in follicle maturation and ovulation were analyzed in follicles grown in vitro. Ar, Pgr, and Has2 mRNA expression levels were the same among the three genotypes. However, βERKO follicles showed reduced expression of Cyp19 mRNA during follicle maturation and reduced Lhcgr and Ptgs2 mRNA expression after human chorionic gonadotropin stimulus. Luteinization occurs normally in αERKO and βERKO follicles, shown by increased progesterone secretion and increased cdkn1b mRNA expression after human chorionic gonadotropin. Collectively, these data indicate that ERβ, but not ERα, plays a direct role in folliculogenesis. ERβ appears to facilitate follicle maturation from the early antral to the preovulatory stage.

The transition from preantral to early antral stage is the penultimate stage of ovarian follicular development in terms of gonadotropin dependence and follicle destiny. Although oocyte-somatic cell communication is important in early follicular development, our knowledge of the precise role of the oocyte-derived growth differentiation factor (GDF)-9 during preantral follicle growth is incomplete. We examined whether and by what means oocyte-derived GDF-9 controls follicular development and steroidogenesis during the preantral to early antral transition, by a combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Intraoocyte injection of GDF-9 antisense suppressed rat preantral follicle growth in vitro, whereas GDF-9 enhanced follicular development. GDF-9 augmented testosterone production in preantral follicles. GDF-9 antisense suppressed androgen production and CYP17A1 mRNA expression in cultured follicles, a response attenuated by exogenous GDF-9. The nonaromatizable androgen 5α-dihydrotestosterone rescued the follicular growth arrest caused by GDF-9 down-regulation. The specific androgen receptor antagonist flutamide suppressed GDF-9-induced preantral follicle growth in vitro. The data suggest that GDF-9 plays an important role in promoting preantral follicle growth by up-regulating follicular androgen biosynthesis. GDF-9 is essential for CYP17A1 expression during follicular development from the preantral to the early antral stage.

Oxygen levels in the follicle are likely to be critical to follicle development. However, a quantitative description of oxygen levels in the follicle is lacking. Mathematical modelling was used to predict the dissolved oxygen levels in the follicular fluid of the developing human follicle. The model predictions showed that follicular fluid dissolved oxygen levels are highly variable among follicles, due to the unique geometry of individual follicles. More generally, predictions showed that oxygen levels in follicular fluid increase rapidly during the initial early antral stages of follicle growth before peaking in the later early antral phase. Follicular fluid dissolved oxygen levels then decline through to the beginning of the pre-ovulatory phase, from which they increase through to ovulation. Based on the best available parameter estimates, the model predictions suggest that the mean dissolved oxygen levels in human follicular fluid during the late antral and pre-ovulatory phases range between 11 and 51 mmHg (~1.5–6.7 vol%). These predictions suggest that the human ovarian follicle is a low-oxygen environment that is often challenged by hypoxia, and are in agreement with only some published data on follicular fluid oxygen levels. Predictions are discussed in relation to follicle health and oocyte culture.

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are known to play key roles in the remodelling of extracellular matrix during ovarian folliculogenesis, especially during the final stage of follicle development. To date, little is known about the significance of MMPs and TIMPs during preantral follicle development. This study determined the expression of MMPs and TIMP-1 during various stages of cat folliculogenesis, largely for the purpose of securing information useful to improving in vitro follicle culture. Primordial (~10 follicles/cat), primary (~5 follicles/cat), secondary (~9 follicles/cat), early antral (~9 follicles/cat), and antral (~4 follicles/cat) follicles were physically isolated from ovaries recovered from 15 cats (5 months to 3 years old during follicular stage) undergoing ovariohysterectomy and assessed for expression of MMP-1, -2, -3, -7, -9, and -13 as well as TIMP-1 using real-time quantitative polymerase chain reaction (q-PCR; 2–4 replicates/follicle stage). Additional ovaries were obtained from three prepubertal (6 months old) and three adult (1 year old) cats and ovarian pieces were fixed in Bouin's solution and assessed for MMP-2 and -13 localization using immunohistochemistry. MMP expression data were analysed using the Kruskull-Wallis one-way ANOVA. Follicles from all stages of development expressed MMPs and TIMP-1. Specifically, expression of MMP-2 increased (P < 0.05) as folliculogenesis progressed (10-fold increases from primordial to early-antral and antral stage). There were no differences (P > 0.05) in the expression of other MMPs among follicular classes. For TIMP-1, there was a tendency (P = 0.07) for increased expression after antrum formation (early antral and antral stages). Immunohistochemistry analysis revealed that MMP-2 was expressed in both the oocyte and somatic cells of all follicular stages in prepubertal cats. However, MMP-2 expression was limited to granulosa and theca cells of antral follicles in adult females. MMP-13 was expressed in the granulosa and theca cells of primary, secondary, and antral stage follicles, and there were no differences (P > 0.05) in localization patterns for this protein between prepubertal and adult females. In summary, the study is the first to report the expression of MMPs as well as TIMP-1 in isolated cat follicles. The difference in MMP-2 expression between prepubertal and adult cats suggests that there may be age-specific requirements for in vitro follicle growth. We are keenly interested in this information for underpinning the development of new in vitro microenvironments for growing immature cat follicles. We suspect that such information will be crucial for understanding how to promote the remodelling of the extracellular matrix by creating degradable biomaterials containing MMP-sensitive peptides to allow optimal follicle expansion.

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand–receptor interactions regarding the transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.

Graphical Abstract Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts. Lay summary The ovary contains hundreds of eggs with only a select few developing from an immature stage through to ovulation over the course of an animal's lifetime. Rescue of eggs from this pool, and the ability to grow them in culture to a mature stage, would be incredibly valuable for fertility preservation efforts in both humans and endangered species. Currently, the isolation of ovarian follicles (eggs with their surrounding helper cells) is a key step in culture systems for large mammalian species, to promote continued growth. Yet, isolation methods may affect the follicle’s future developmental capacity. We evaluated two isolation strategies, mechanical micro-dissection (needle/scalpel blade) and enzymatic digestion (using Liberase blendzyme) on ovaries of domestic cats obtained via routine spay procedures. Mechanically isolated follicles displayed improved growth, survival, and indications of developmental competence in 14-day culture, compared with high concentration (1.4 Wünsch units/mL) enzyme-isolated follicles. However, mechanical isolation was not different from low (0.7 Wünsch units/mL) enzyme for these metrics, or for expression of key genes indicative of follicular cell functions. Further, differences in follicle growth/survival were not apparent until 7+ days in culture. Thus, ovarian follicle isolation strategies influence developmental potential in culture, and extended culture will be required to identify optimal methods for fertility preservation efforts.

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1–3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3–4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1–3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

SummaryThe aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

Fertility preservation has received unprecedented attention nowadays. In addition to cryopreservation and re-implantation of embryos, oocytes, and ovarian tissue pieces, in vitro culture system for follicles/oocytes has been considered an alternative strategy for fertility preservation. Reproduction strategies based on the recovery of oocytes' population from antral follicles are unsatisfactory, and the success of this approach has not exceeded 35% of embryos produced in vitro for over 30 years. The possibility of accessing the reserve of smaller follicles (primordial, secondary, and up to the preantral stage) would amplify the number of gametes available for increasing reproductive potential. Furthermore, this would open enormous prospects for the rescue of fertility in various conditions in the human clinic and genetic rescue in animal breeding and biodiversity preservation programs. However, this would require developing protocols capable of growing immature oocytes to the stage in which they can be matured and fertilized in vitro. Culture systems to achieve in vitro growth (IVG) of immature oocytes to maturity and subsequent fertilization in vitro (IVF) have been the subject of research for almost 40 years. Several systems that support the growth of later stages of follicle development from rodents have been developed, with some reporting the production of live young, but they are still at an experimental stage, and further research is required before the protocols could be clinically applied. One of the significant limitations is identifying growth factors, hormones, and nutrients necessary for each specific follicle development stage. This evidence has led to hypothesize the development of culture systems consisting of a step-by-step approach, although no reliable protocols have been developed so far. The oocyte culture at the early stages of development represents an alternative to maximize the potential source of gamete used for fertility preservation. Several attempts have been made to recreate these conditions in vitro, but no reliable protocols have been developed to date. The lack of knowledge in the mechanisms involved in the early development of the oocyte and this passage from growing to fully grown stage be one of the most critical steps during oocyte development, these still represent the significant limiting factor for this technology. The studies conducted during the doctorate program led to defining a physiological culture system that successfully differentiated growing bovine oocytes. This study used parameters predictive of oocyte differentiation to evaluate the current technique's efficiency and efficacy. Based on previous observations from our laboratory, we initially hypothesized that zinc plays a role during the latest stages of oocyte growth and differentiation, particularly in controlling transcription during the final stage of oocyte growth. This first study demonstrated that zinc supplementation improves the meiotic competence of growing oocytes, affects the global transcription activity and the global DNA methylation. This information was used in the next part to better define a culture system for growing oocytes. The subsequent study provided a 5-days protocol named L-IVCO (long in vitro culture of oocytes) to promote growing oocyte differentiation until the acquisition of meiotic and embryonic developmental competencies in a significantly higher proportion of the published protocols. This study demonstrated that a physiological medium could support a gradual transition of the oocyte from immature to mature stage, thus generating suitably quality blastocysts after fertilization. In conclusion, our studies provide an improved protocol that can increase the source of fertilizable gametes in preservation programs and gives a prospective approach in human clinics, animal breeding programs, and salvage intervention of threatened species. Moreover, our studies defined a model to perform in-depth studies of the cellular and molecular processes that regulate the acquisition of meiotic and developmental competence during oocyte differentiation.

This chapter discusses the varying changes in female hormones that begin 20 to 30 years prior to a woman’s final menstrual period (FMP), commonly around 50 years of age. Both testosterone and progesterone begin declining in a woman’s 20s and 30s, respectively, and antral follicle count in her ovaries also declines. In contrast, estrogen levels remain relatively stable from puberty until a woman’s early 40s This chapter discusses the changes that occur in a woman’s sexual health and overall health and well-being as her hormones change throughout her lifespan. It also discusses the controversy and misleading information about conventional hormone replacement therapies and presents case examples of postmenopausal women’s sexual health and overall health and well-being being restored with an integrative approach using custom-compounded bioidentical hormone replacement. A relatively new (nonsurgical) radiofrequency treatment for vulvo-vaginal rejuvenation for vaginal atrophy and urinary leakage problems in older women is also discussed.