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Mary F. Hay, R. M. Moor, D. G. Cran and H. M. Dott

Summary. Large (4–6 mm diam.) and small (2–3 mm) atretic follicles were removed from sheep ovaries during the luteal phase of the cycle and maintained in organ culture without hormonal supplementation for up to 5 days. The structure, cell dynamics and steroid-producing capacity of the follicles were compared with those of non-atretic follicles of similar size.

The granulosa layer of the atretic follicles invariably regenerated in culture, increasing in thickness more than 2- and 4-fold in large and small follicles respectively. This could not be accounted for by cell division which remained low throughout the culture period. In contrast, non-atretic follicles showed high mitotic activity during the first 24 h in culture: this was not associated with an increase in granulosa thickness in large follicles although there was a 4-fold increase in small ones. An increase in internuclear spacing, a measure of cell size plus intercellular space, partly accounted for the increase in granulosa thickness in atretic follicles. Even when granulosa cells remained in close apposition there was an almost total absence of gap junctions, a prominent feature in the granulosa of non-atretic follicles both in vivo and in vitro. Pyknotic nuclei and atretic bodies rapidly disappeared from the regenerating granulosa layer. The theca interna was restored in culture to a state ultrastructurally closely resembling that of non-atretic follicles in vivo.

Total steroid secretion (oestradiol-17β, testosterone plus progesterone) into the culture medium ( tissue−1.24 h−1) was the same for atretic and non-atretic follicles of comparable size. There was, however, a marked difference in the type of steroid produced, largely related to a loss of aromatizing capacity in atretic follicles. The predominant steroid secreted by large non-atretic follicles was osetrogen, with slightly smaller amounts of testosterone, whereas the principal steroid secreted by large atretic follicles was progesterone. In small non-atretic and atretic follicles, the predominant steroid was testosterone, but the non-atretic follicles also secreted appreciable amounts of oestrogen. Addition of FSH to the culture medium did not restore aromatizing capacity to the atretic follicles.

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A. Fehrenbach, N. Nüsse and P. L. Nayudu

This study investigated the relationship between individual follicle growth, steroid release and follicular morphology to provide basic information about critical stages in follicle development. Preantral mouse follicles secreted significant and constant amounts of progesterone that were not related to oestradiol production but did appear to be related to thecal organization. Oestradiol release was variable among follicles of equivalent diameter, but marked increases in oestradiol concentrations were measured in follicles of 300 and 400 μm diameter. Over 4 days of culture, the proportion of follicles growing beyond the threshold diameter of 300 μm was lower in small (140–160 μm) than it was in large preantral follicles (170–210 μm). Retardation of growth below this diameter was associated with significantly decreased steroid concentrations. In follicles growing beyond the threshold diameter, antrum formation progressed, but full Graafian stage was not attained. Among large follicles, variability in oestradiol release could not be associated with obvious histological differences. In contrast, structural disturbances were associated with reduced steroid production and growth abnormalities. Therefore, it is concluded that the preantral follicles selected for culture comprise distinct follicle populations with respect to their developmental potential in vitro, and that follicle development is controlled by endogenous processes involving oestradiol at distinct stages of follicle development.

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Paul A Fowler and Norah Spears

Gonadotrophin surge-attenuating factor (GnSAF) bioactivity (the suppression of GnRH-induced but not basal LH and FSH secretion from pituitary gonadotrophs) is produced by granulosa cells in vitro. Previous studies to investigate this bioactivity used dispersed granulosa cells which lack some cell types and the structural components of the follicle in vivo. The aim of this study, therefore, was to investigate whether intact rodent follicle culture was a suitable model for the study of the production of GnSAF bioactivity, allowing GnSAF to be investigated in a more physiologically realistic environment while still retaining culture conditions from which, as with granulosa cell cultures, extraneous factors can be excluded. Follicles from 16-day-old rats and 21-day-old mice were cultured for 3–6 days in the presence or absence of FSH and/or LH. The follicle-conditioned medium, and matching samples of unconditioned culture medium were added to our established rat pituitary monolayer GnSAF bioassay. Both mouse and rat intact follicles produced GnSAF bioactivity, reducing GnRH-induced LH secretion significantly. GnSAF output from the mouse follicles was highest during days 1–3 of culture, when follicles were at an early antral stage of development, and fell on days 4–6 as the follicles grew to the mid antral stage. While the stimulatory effects of FSH on rat follicle GnSAF secretion was dose-dependent, LH alone did not increase GnSAF production. An antibody against human GnSAF blocked GnSAF bioactivity produced by rat follicles, and recognised proteins within the expected pI and molecular weight range for GnSAF in two-dimensional gels of rat follicle-conditioned medium, showing a good homology between rodent and human GnSAF proteins. In conclusion, the release of GnSAF bioactivity is principally from small follicles stimulated by FSH. Therefore, intact rodent follicle culture systems offer an excellent model for the investigation of factors controlling GnSAF production under relatively physiological conditions.

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Elizabeth M Parrish, Anaar Siletz, Min Xu, Teresa K Woodruff and Lonnie D Shea

Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell–cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus–oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.

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K. M. Henderson, K. P. McNatty, R. L. Wards, D. A. Heath and S. Lun

Summary. The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10–13 of the oestrous cycle, from Booroola × Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3–4·5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P < 0·05) than that by cells from non-atretic 3–4·5 mm diameter ++ follicles, but similar to that of cells from non-atretic ⩾5 mm diameter ++ follicles. The production of oestradiol-17β by cells cultured in the presence of testosterone (1 μg/ml followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17β production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17β synthesis, testosterone generally had a slight, stimulatory effect on inhibin production. Irrespective of follicle size, or genotype, no detectable amounts of inhibin were produced by granulosa cells from atretic follicles during the first 24 h of culture, or by cells from nonatretic or atretic follicles during the second 24 h of culture.

These studies show that the highest mean amounts of inhibin produced by granulosa cells in vitro are similar for both genotypes. Moreover, they are achieved with cells from the largest nonatretic follicles in both ++ (i.e. ⩾5 mm diameter) and FF (i.e. 3–4·5 mm diameter) ewes.

Keywords: Booroola ewes; inhibin; granulosa cells; oestradiol-17β

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J. Testart, A. Thébault and Brigitte Lefèvre

Summary. Follicular rupture was never observed when preovulatory rabbit follicles were isolated from the ovary before the ovulatory gonadotrophin discharge and placed in hormone-free culture for 10–14 h (n = 48). However, if the follicles were taken 1 h post coitum and cultured under the same conditions, 11/24 (45·8%) ruptured spontaneously. Follicular rupture under these conditions appeared to be chronologically and histologically comparable to that of ovulation in vivo. Culture in the presence of progesterone increased the proportion of follicles ovulating in vitro (17/23 = 73·9%; P < 0·05). Inhibition in vitro of the synthesis of steroids or prostaglandins suppressed ovulation, although meiotic maturation of the oocyte did take place. These findings indicate that the follicle constitutes an independent entity within the ovary from 1 h post coitum.

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Mammalian ovarian oocytes can mature in vitro when liberated from Graafian follicles and placed in appropriate culture media. This was first demonstrated in the rabbit (Pincus & Enzmann, 1935, 1937; Chang, 1955a, b) and has been confirmed in a variety of animals (see Donahue, 1972; Biggers, 1973; Fowler & Edwards, 1973).

According to Jagiello (1969), 80 to 100% of guinea-pig oocytes isolated from ovaries of adult females in the middle (Days 5 to 8) of their oestrous cycle resume meiosis upon culture and may reach metaphase II by 14 hr of culture. Unfortunately, Jagiello gave no detailed information of the techniques which she used for isolation and culture of the oocytes and did not determine whether the oocytes matured in vitro were fertilizable. This paper describes a method of culturing

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Sarah E Harris, Iris Adriaens, Henry J Leese, Roger G Gosden and Helen M Picton

Metabolic markers are potentially valuable for assessment of follicle development in vitro. Carbohydrate metabolism of murine preantral follicles grown to maturityover 13 days in vitro has been measured, and metabolism of resulting oocyte–cumulus complexes (OCCs) and denuded oocytes has been compared with in vivo ovulated control counterparts. Spent follicle culture media were analysed for glucose, lactate and pyruvate concentrations. During follicle in vitro growth, glycolysis accounted for a rise from ∼24 to 60% of all glucose consumed. Ovulation induction caused a significant increase in glucose uptake and lactate production by in vitro-grown follicles to 71.7±1.2 and 96.6±4.8 nmoles/day respectively. OCCs grown in vitro had significantly higher rates of glucose consumption and lactate and pyruvate production (110.1± 3.5, 191.8± 8.9 and 31.7± 1.7 pmoles/h respectively) than in vivo ovulated controls (67.4± 8.1, 113.9± 17.1 and 20.2± 4.0 pmoles/h respectively), but a reduced capacity for pyruvate consumption (1.13± 0.06 vs 1.49± 0.06 pmoles/h by in vivo ovulated oocytes). Metabolism of OCCs was affected by the quality of the original follicle. In vitro-grown oocytes had a reduced cytoplasmic volume when compared with controls (168.3± 2.0 vs 199.0± 3.2 proportionately respectively) but a similar rate of metabolism per unit volume. Meiotic status influenced metabolism of both OCCs and denuded oocytes. In conclusion, glucose consumption and lactate production by cultured follicles increased in tandem with developmental progression and were stimulated prior to ovulation. Additionally, the metabolic profiles of in vitro produced OCCs and the oocytes within them are affected by long-term exposure to the culture environment.

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Isam B Sharum, Sofia Granados-Aparici, Fiona C Warrander, Felicity P Tournant and Mark A Fenwick

The molecular mechanisms involved in regulating the development of small, gonadotrophin-independent follicles are poorly understood; however, many studies have highlighted an essential role for TGFB ligands. Canonical TGFB signalling is dependent upon intracellular SMAD proteins that regulate transcription. STRAP has been identified in other tissues as an inhibitor of the TGFB–SMAD signalling pathway. Therefore, in this study we aimed to determine the expression and role of STRAP in the context of early follicle development. Using qPCR, Strap, Smad3 and Smad7 revealed similar expression profiles in immature ovaries from mice aged 4–16 days containing different populations of early growing follicles. STRAP and SMAD2/3 proteins co-localised in granulosa cells of small follicles using immunofluorescence. Using an established culture model, neonatal mouse ovary fragments with a high density of small non-growing follicles were used to examine the effects of Strap knockdown using siRNA and STRAP protein inhibition by immuno-neutralisation. Both interventions caused a reduction in the proportion of small, non-growing follicles and an increase in the proportion and size of growing follicles in comparison to untreated controls, suggesting inhibition of STRAP facilitates follicle activation. Recombinant STRAP protein had no effect on small, non-growing follicles, but increased the mean oocyte size of growing follicles in the neonatal ovary model and also promoted the growth of isolated preantral follicles in vitro. Overall findings indicate STRAP is expressed in the mouse ovary and is capable of regulating development of small follicles in a stage-dependent manner.

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R. M. Moor and A. O. Trounson

Summary. Oocytes removed from, or retained within, non-atretic and atretic follicles of different sizes were cultured for 24 h in the presence of a variety of hormones in an attempt to identify the factors affecting oocyte maturation in vitro. Resumption of meiosis was assessed morphologically; the developmental capacity of oocytes after culture was determined by transfer to the oviducts of inseminated ewes.

About 70% of oocytes cultured after removal from follicles of different sizes resumed meiosis in vitro, but they did not undergo normal development after transplantation.

Oocytes cultured within the follicle in hormone-free medium remained at the germinal vesicle stage. In the presence of FSH and LH some oocytes reached the second meiotic metaphase: 19% in small (2–3 mm diam.) and 73% in larger (3–5 mm diam.) non-atretic follicles, and 54% in small and 45% in larger atretic follicles.

Less than 5% of oocytes cultured in follicles developed into normal blastocysts after transplantation when either no hormone or only FSH and LH were added to the culture medium. The addition of oestradiol-17β to medium containing FSH (2 μg/ml) and LH (1 μg/ml) resulted in the development to blastocysts of 26% of oocytes from small non-atretic follicles, 46% from large non-atretic follicles and 50% from atretic follicles. Blastocyst formation was greatly depressed and fragmentation rate significantly increased with concentrations of 10 μg FSH/ml and 2 μg LH/ml.

Developmental capacity after culture was further demonstrated by the birth of lambs from 63% of blastocysts derived from oocytes matured in vitro; 52% of control blastocysts developed to lambs after transfer.