A serum-free ovine granulosa cell culture system is described that allows the induction of FSH-responsive oestradiol production by undifferentiated cells from small (< 3.5 mm) follicles (P < 0.001) and the maintenance of oestradiol production by differentiated cells from large (≥3.5 mm) follicles. Physiological doses of FSH stimulated (P < 0.01) proliferation of cultured granulosa cells from both small and large follicles. The synthesis of immunoreactive inhibin and progesterone by granulosa cells from small and large follicles increased (P < 0.01) with time of culture, and was not dependent on FSH. Inhibin secretion expressed on a per cell basis was not FSH responsive. Insulin and insulin-like growth factor I (IGF-I), in the presence of FSH, stimulated (P < 0.001) cell proliferation and oestradiol and inhibin production by granulosa cells from small and large follicles. There was a significant (P < 0.001) interaction between insulin and IGF-I in the stimulation of granulosa cell proliferation and differentiation. Both epidermal growth factor (EGF) and transforming growth factor α (TGF-α) in the presence of FSH stimulated cellular proliferation (P < 0.001) in a dose-responsive manner and concomitantly inhibited (P < 0.001) oestradiol and inhibin secretion. The development of this granulosa cell culture system will make it possible to study, in vitro, the cascade of events that controls granulosa cell differentiation and ultimately follicle selection in sheep.
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B. K. Campbell, R. J. Scaramuzzi and R. Webb
HM Knijn, C Wrenzycki, PJ Hendriksen, PL Vos, D Herrmann, GC van der Weijden, H Niemann and SJ Dieleman
Bovine embryos produced in vitro differ substantially from embryos produced in vivo in the mRNA expression patterns of genes important for development. Several factors in the in vitro production systems have profound effects on embryonic mRNA expression patterns. The effects of the type of maturation on the expression pattern of genes important for development in blastocysts produced in vitro have not yet been investigated. The aim of the present study was to investigate the effects of various maturational protocols on the relative abundance of a panel of six marker genes, indicative of compaction and cavitation, metabolism, stress susceptibility and RNA processing, in bovine blastocysts produced in vitro. Four groups of blastocysts were analysed by a sensitive semi-quantitative RT-PCR assay. Blastocysts were produced in vitro from oocytes of different origin from: (1) 3-8 mm follicles; (2) preovulatory follicles before the LH surge; and (3) preovulatory follicles 24 h after the LH surge. The first two groups were matured in vitro, whereas the third group had undergone maturation in vivo. A fourth group comprised blastocysts developed entirely in vivo. Expression of glucose transporter 1 was significantly (P < 0.05) higher, and expression of desmocollin 2 and plakophilin tended to be higher (P < 0.1) for in vivo (group 4) compared with in vitro blastocysts (group 1), whereas no differences were found for heat shock protein 70.1, E-cadherin and poly(A) polymerase. Expression of the six transcripts did not differ among blastocysts produced in vitro from oocytes of groups 1, 2 and 3. Results indicate that alterations in the relative abundance of these transcripts in blastocysts produced in vitro cannot primarily be attributed to the origin of the oocyte, but are likely to have been induced by post-maturation or fertilization culture conditions.
Mehmet Uzumcu, Zui Pan, Yi Chu, Peter E Kuhn and Rob Zachow
Hepatocyte growth factor (HGF) regulates granulosa cell (GC) steroidogenesis and suppresses apoptosis in non-ovarian cells. The hypothesis was thus developed that intraovarian HGF supports folliculogenesis by mediating steroidogenesis and suppressing apoptosis. To investigate the latter, the anti-apoptotic actions of HGF were tested in GCs and follicles isolated from immature rats. Results showed that HGF suppressed apoptosis in GC and follicle cultures as visualized using apoptosis indicator dye, YO-PRO-1. Immunohistochemistry was used to investigate the distribution of HGF, c-met, and HGF activator (HGFA) protein during folliculogenesis in equine chorionic gonadotropin (eCG)-primed rats. Immunoreactive HGF content was the greatest in GCs within preantral follicles. Following eCG, large antral follicles showed elevated HGF staining in theca and interstitial cells when compared with GCs. Intense c-met staining was observed in GCs within non-primed small preantral follicles; following eCG, the level of c-met was diminished in GCs, but increased within theca and interstitial cells. Theca, interstitium, and GCs in non-primed and primed ovaries contained HGFA. Following eCG, HGFA was more apparent in theca cells and the interstitium when compared to that in GCs within large antral follicles. The presence of HGF, c-met, and HGFA in preantral follicles would potentially enable the anti-apoptotic effects of HGF that were observed in vitro to occur in vivo. Advanced folliculogenesis led to a change in the cellular distribution of the HGF, c-met, and HGFA, suggesting that the ovarian HGF system is hormonally regulated in vivo.
EC dela Pena, Y Takahashi, S Katagiri, EC Atabay and M Nagano
Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.
M Sakaguchi, T Dominko, N Yamauchi, ML Leibfried-Rutledge, T Nagai and NL First
The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.
DG Armstrong, JG Gong, JO Gardner, G Baxter, CO Hogg and R Webb
The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-I) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford x Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period (17 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-I or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.
D. T. Baird
After autotransplantation of different cell types to the anterior chamber of the eye in the rat, Falck (1959) proposed that granulosa and theca interna cells complemented one another in the synthesis of oestrogen by the Graafian follicle. Although numerous experiments in vitro support this 'two cell' hypothesis (Ryan, Petro & Kaiser, 1968; Makris & Ryan, 1975), most evidence suggests that in vivo oestrogen is synthesized exclusively by the theca cells (YoungLai & Short, 1970; Channing & Coudert, 1976). However, the report that FSH stimulates specifically the aromatization of androgens to oestrogens by ovaries in organ culture (Moon, Dorrington & Armstrong, 1975), as well as by granulosa cells in tissue culture (Dorrington, Moon & Armstrong, 1975), led us to re-examine the biosynthesis of oestrogens by the Graafian follicle in vivo. If androgens leave the thecal cell before aromatization, it should be possible to make them unavailable for the synthesis of oestrogen by antiandrogen antiserum.
Summary. Female mice of the KE and CBA strains were used to examine the rate of oocyte maturation in vivo and in vitro. In CBA females killed just before ovulation most preovulatory oocytes were already in the metaphase II stage, while the oocytes of KE mice were arrested at metaphase I until the time of ovulation, and further stages of maturation occurred in the oviduct, reaching the metaphase II stage 3–5 h later. A similar strain difference in oocyte maturation rate was observed from in-vitro culture of cumulus-free oocytes, isolated from the ovaries of PMSG-primed females and intact females killed at the metoestrous phase of the cycle. This indicates that the strain-specific course of maturation is determined in the oocyte by a few days before ovulation. Therefore, if the rate of oocyte maturation is influenced by somatic components of the follicle, this must occur at some earlier stages of follicle development.
Song Li, Qi Fan, Yanqiu Xie, Haiyan Lin, Qi Qiu, Yihua Liang and Qingxue Zhang
In vitro activation of primordial follicles is becoming more essential in assisted reproductive technologies. Vasoactive intestinal peptide (VIP) is one of the members of the neurotrophin family which has demonstrated to have an impact on follicle development in recent years. This study aims to investigate the effect of VIP on the activation of primordial follicles in neonatal rat in an in vitro culture system and to determine the relevant molecular mechanism of their activation. Ovaries of 4-day-old rats were examined for the expression of VIP receptors and were cultured in mediums containing VIP with or without inhibitors of the ERK–mTOR signalling pathway. They were then collected for histological analysis or measurement of the molecular expression of this pathway. The receptors of VIP were found in granular cells and oocytes of primordial and early-growing follicles in neonatal ovary. The ratio of growing follicle increased in the presence VIP at different concentrations, with the highest level of increase being observed in the 10−7 mol/L VIP-treated group. The ratio of PCNA-positive granular cells was also increased, while that of the apoptotic oocytes were decreased, and protein analysis showed increased phosphorylation of ERK1/2, mTOR and RPS6 in the VIP-treated group. However, the effect of VIP on the activation of primordial follicle became insignificant with the addition of MEK inhibitor (U0126) or mTORC1 inhibitor (rapamycin). This study indicated that VIP could activate neonatal rat primordial follicle through the ERK-mTOR signalling pathway, suggesting a strategy for in vitro primordial follicle recruitment.
MARY F. HAY and R. M. MOOR
The localization of Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD) has been examined in ovarian follicles in vivo and in vitro, and related to oestrogen and progesterone production.
In vivo, during the oestrous cycle, enzyme activity was restricted to the theca interna of the one or two most advanced follicles in each animal, but was present only between Days 2 and 5 and between Day 13 and ovulation. High levels of oestrogen were found in the ovarian venous blood only when follicles containing 3β-HSD were present. When sheep were injected with PMSG, the theca interna in a number of small follicles acquired 3β-HSD activity and began to secrete oestrogen within 12 hr of the injection. The enzyme was not detected in the membrana granulosa of any follicles before ovulation but within a few hours of ovulation, 3β-HSD activity was present in the granulosa lutein cells.
In vitro, large activated follicles exhibited 3β-HSD activity in the theca interna and secreted high levels of oestrogen into the culture medium. When LH was added to the medium, oestrogen secretion was inhibited; within 48 hr, the follicles were secreting high levels of progesterone, and 3β-HSD activity was present in both the membrana granulosa and the theca interna. Dibutyryl cyclic adenosine monophosphate mimicked the effect of LH in suppressing oestrogen secretion, but did not induce production of progesterone; the distribution of 3β-HSD activity in follicles treated with this nucleotide was the same as in those cultured in control medium.