Growth Of Ovarian Antral Follicles Research Articles

Imidacloprid and Its Bioactive Metabolite, Desnitro-Imidacloprid, Differentially Affect Ovarian Antral Follicle Growth, Morphology, and Hormone Synthesis In Vitro.

Imidacloprid is a neonicotinoid pesticide used in large-scale agricultural systems, home gardens, and veterinary pharmaceuticals. Imidacloprid is a small molecule that is more water-soluble than other insecticides, increasing the likelihood of large-scale environmental accumulation and chronic exposure of non-targeted species. Imidacloprid can be converted to the bioactive metabolite desnitro-imidacloprid in the environment and body. Little is known about the mechanisms by which imidacloprid and desnitro-imidacloprid induce ovarian toxicity. Thus, we tested the hypothesis that imidacloprid and desnitro-imidacloprid differentially affect antral follicle growth and steroidogenesis in vitro. Antral follicles were dissected from the ovaries of CD-1 mice and cultured in media containing vehicle control or 0.2 µg/mL-200 µg/mL of imidacloprid or desnitro-imidacloprid for 96 h. Follicle morphology was monitored, and follicle size was measured every 24 h. At the end of the culture periods, media were used to quantify follicular hormone levels, and follicles were used for gene expression analysis of steroidogenic regulators, hormone receptors, and apoptotic factors. Imidacloprid did not affect follicle growth or morphology compared to the control. Desnitro-imidacloprid inhibited follicle growth and caused follicles to rupture in culture compared to the control. Imidacloprid increased progesterone, whereas desnitro-imidacloprid decreased testosterone and progesterone compared to the control. Desnitro-imidacloprid also changed estradiol compared to the control. At 48 h, IMI decreased the expression of Star, Cyp17a1, Hsd17b1, Cyp19a1, and Esr2 and increased the expression of Cyp11a1, Cyp19a1, Bax, and Bcl2 compared to the control. IMI also changed the expression of Esr1 compared to the control. At 48 h, DNI decreased the expression of Cyp11a1, Cyp17a1, Hsd3b1, Cyp19a1, and Esr1 and increased the expression of Cyp11a1, Hsd3b1, and Bax compared to the control. At 72 h of culture, IMI significantly decreased the expression of Cyp19a1 and increased the expression of Star and Hsd17b1 compared to the control. At 72 h, DNI significantly decreased the expression of Cyp11a1, Cyp17a1, Hsd3b1, and Bax and increased the expression of Esr1 and Esr2. At 96 h, IMI decreased the expression of Hsd3b1, Cyp19a1, Esr1, Bax, and Bcl2 compared to the control. At 96 h, DNI decreased the expression of Cyp17a1, Bax, and Bcl2 and increased the expression of Cyp11a1, Hsd3b1, and Bax compared to the control. Together, these data suggest mouse antral follicles are targets of neonicotinoid toxicity, and the mechanisms of toxicity differ between parent compounds and metabolites.

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

Effects of in vitro exposure to dibutyl phthalate, mono-butyl phthalate, and acetyl tributyl citrate on ovarian antral follicle growth and viability.

Dibutyl phthalate (DBP) is present in consumer products and the coating of some oral medications. Acetyl tributyl citrate (ATBC) has been proposed as an alternative to DBP because DBP causes endocrine disruption in animal models. Following ingestion, DBP is converted to its main metabolite mono-butyl phthalate (MBP) which has been detected in >90% of human follicular fluid samples. Previous studies show that DBP reduces the number of antral follicles present in the ovaries of mice. Thus, this study was designed to evaluate the effects of DBP, MBP, and ATBC on in vitro growth and viability of mouse ovarian antral follicles. Antral follicles were isolated from CD-1 females (PND32-37) and treated with vehicle, DBP, MBP, or ATBC (starting at 0.001 and up to 1000 μg/ml for DBP; 24-72 h). Follicle diameter, ATP production, qPCR, and TUNEL were used to measure follicle growth, viability, cell cycle and apoptosis gene expression, and cell death-associated DNA fragmentation, respectively. While MBP did not cause toxicity, DBP exposure at ≥10 μg/ml resulted in growth inhibition followed by cytoxicity at ≥500 μg/ml. ATBC increased the number of nongrowing follicles at 0.01 μg/ml and did not affect ATP production, but increased TUNEL positive area in treated follicles. Gene expression results suggest that cytotoxicity in DBP-treated follicles occurs via activation of cell cycle arrest prior to follicular death. These findings suggest that concentrations of DBP ≥10 μg/ml are detrimental to antral follicles and that ATBC should be examined further as it may disrupt antral follicle function at low concentrations.

Effects of isoliquiritigenin on ovarian antral follicle growth and steroidogenesis

Isoliquiritigenin is a botanical estrogen used as a dietary supplement. Previous studies show that other botanical estrogens affect ovarian estradiol synthesis, but isoliquiritigenin’s effects on the ovary are unknown. Thus, this study tested the hypothesis that isoliquiritigenin inhibits ovarian antral follicle growth and steroidogenesis. Antral follicles from CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or isoliquiritigenin (0.6μM, 6 μM, 36 μM, and 100 μM) for 48–96h. During culture, follicle diameters were measured daily to assess follicle growth. After culture, media were collected for hormone assays and follicles were collected for gene expression analysis of steroidogenic enzymes. Isoliquiritigenin inhibited antral follicle growth and altered estradiol, testosterone, and progesterone levels. Additionally, isoliquiritigenin altered the mRNA levels of cytochrome P450 steroid 17-α-hydroxylase 1, aromatase, 17β-hydroxysteroid dehydrogenase 1, and steroidogenic acute regulatory protein. These data indicate that exposure to isoliquiritigenin inhibits growth and disrupts steroid production in antral follicles.

Temephos decreases ovarian antral follicle growth in an in vitro system

Temephos decreases ovarian antral follicle growth in an in vitro system

In vitro re-expression of the aryl hydrocarbon receptor (Ahr) in cultured Ahr-deficient mouse antral follicles partially restores the phenotype to that of cultured wild-type mouse follicles

BackgroundThe aryl hydrocarbon receptor (AHR) mediates the toxic effects of various endocrine disrupting chemicals. In female mice, global deletion of the Ahr (AhrKO) results in slow growth of ovarian antral follicles. No studies, however, have examined whether injection of the Ahr restores the phenotypes of cultured AhrKO ovarian antral follicles to wild-type levels. MethodsWe developed a system to construct a recombinant adenovirus containing the Ahr to re-express the Ahr in AhrKO granulosa cells and whole antral follicles. We then compared follicle growth and levels of factors in the AHR signaling pathway (Ahr, Ahrr, Cyp1a1, and Cyp1b1) in wild-type, AhrKO, and Ahr re-expressed follicles. Further, we compared the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild-type, AhrKO, and Ahr re-expressed follicles. ResultsAhr injection into AhrKO follicles partially restored their growth pattern to wild-type levels. Further, Ahr re-expressed follicles had significantly higher levels of Ahr, Ahrr, Cyp1a1, and Cyp1b1 compared to wild-type follicles. Upon TCDD treatment, only Cyp1a1 levels were significantly higher in Ahr re-expressed follicles compared to the levels in wild-type follicles. ConclusionOur system of re-expression of the Ahr partially restores follicle growth and transcript levels of factors in the AHR signaling pathway to wild-type levels.

Granulosa Cells from Emerged Antral Follicles of the Bovine Ovary Initiate Inflammation in Response to Bacterial Pathogen-Associated Molecular Patterns via Toll-Like Receptor Pathways1

Bacterial infections of the uterus or mammary gland commonly perturb ovarian antral follicle growth and function, causing infertility in cattle. Cells of the innate immune system use Toll-like receptors (TLRs) TLR2, TLR4, and TLR5 to recognize pathogen-associated molecular patterns (PAMPs) of bacteria, leading to production of inflammatory mediators, such as IL-1beta, IL-6, and IL-8. The present study examined whether granulosa cells from emerged antral follicles have functional responses to typical bacterial PAMPs. Granulosa cells from emerged bovine antral follicles expressed mRNA for all 10 TLRs. Cellular expression of mRNA for the cytokines IL1B, IL6, IL10, and TNF, and chemokines IL8 and CCL5, increased after treatment with synthetic bacterial lipoprotein binding TLR2, lipopolysaccharide binding TLR4, or flagellin binding TLR5. Supernatants of granulosa cells accumulated IL-1beta, IL-6, and IL-8 protein in a concentration-dependent manner when treated with lipoprotein or lipopolysaccharide, but not flagellin. Accumulation of IL6 in response to lipoprotein and lipopolysaccharide was attenuated using siRNA targeting TLR2 and TLR4, respectively. Granulosa cells increased phosphorylation of mitogen-activated protein kinase (MAPK) 14 and MAPK3/1 within 30 min of treatment with lipopolysaccharide or lipoprotein, and inhibitors targeting MAPK14 reduced the accumulation of IL-6 in response to the PAMPs. Treatment with hormones follicle-stimulating hormone, luteinizing hormone, estradiol, or progesterone did not significantly affect granulosa cell responses to PAMPs. However, epidermal growth factor enhanced IL-6 accumulation in response to lipoprotein and inhibiting epidermal growth factor receptor (EGFR) abrogated the effect, whereas lipoprotein increased granulosa cell EGFR mRNA expression. In conclusion, bovine granulosa cells from emerged follicles sense bacterial PAMPs and initiate inflammatory responses via TLR2 and TLR4 pathways.

Mono-(2-ethylhexyl) Phthalate Induces Oxidative Stress and Inhibits Growth of Ovarian Antral Follicles.

Phthalates are synthetic plasticizers that are widely used in plastics and many other common consumer products. Exposure to these chemicals can cause developmental and reproductive toxicity. Our previous studies show that di-(2-ethylhexyl) phthalate (DEHP) inhibits follicle growth by inducing oxidative stress. In this study, we tested the hypothesis that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, also inhibits the growth of antral follicles through an oxidative stress pathway. Antral follicles were isolated from ovaries of adult CD-1 mice (age 30-35 days) and cultured with vehicle control (dimethylsulfoxide, DMSO) or MEHP (0.1-100μg/ml) ± 0.5-1mM N-acetyl cysteine (NAC, an antioxidant) for 96h. During culture, follicle size was evaluated daily as a measurement of follicle growth. At the end of the culture, follicles were collected and processed for in vitro reactive oxygen species (ROS) assays, which measure the presence of free radicals in the samples, an indicator of oxidative stress. The results indicate that MEHP suppresses the growth of follicles and increases ROS levels compared to DMSO controls, whereas NAC blocks the ability of MEHP to inhibit follicle growth and protects the follicles from MEHP-induced ROS levels. To further analyze the mechanism by which MEHP causes oxidative stress and inhibits follicle growth, we measured the expression and activity of various key antioxidant enzymes: Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT). The results suggest that MEHP does not affect Cat expression and that it only decreases Sod1 and Gpx expression at MEHP 100 μg/ml, but not at lower doses. The results from enzyme activity assays indicate that MEHP inhibits GPX activity, but increases CAT activity at 0.1 and 10μg/ml. Interestingly, MEHP increases SOD1 activity at 10μg/ml, but inhibits SOD1 activity at 100μg/ml. Furthermore, NAC blocks the effects of MEHP (100μg/ml) on the expression and activity of SOD1 and GPX. Collectively, these data suggest that MEHP inhibits follicle growth by inducing production of ROS and by altering the expression and activity of key antioxidant enzymes. Supported by: NIHES019178 (JAF), Billie A Field Fellowships (WW and ZRC), and the Environmental Toxicology Scholar Program (MSB).

Bisphenol A Targets Atresia Factors and Cell Cycle Regulators to Inhibit Ovarian Antral Follicle Growth.

Bisphenol A Targets Atresia Factors and Cell Cycle Regulators to Inhibit Ovarian Antral Follicle Growth.

Di-(2-ethylhexyl) Phthalate Inhibits Growth of Ovarian Antral Follicles Through an Oxidative Stress Pathway.

Di-(2-ethylhexyl) Phthalate Inhibits Growth of Ovarian Antral Follicles Through an Oxidative Stress Pathway.

Effects of estradiol and progesterone on circulating LH and FSH secretion, and ovarian antral follicle growth in anestrous ewes

In the ewe, ovarian antral follicles emerge or grow in a wave-like pattern and each wave is preceded by a peak in the serum FSH level. The purpose of the current study was to investigate whether in anestrous Western White Face ewes, a combination of progesterone and estradiol affects the circulating FSH peak secretion and the number of small ovarian follicles. Five ewes were treated with subcutaneous silastic rubber implants (10 cm × 0.47 cm), containing 10% estradiol-17β w/w (controls) and 5 ewes were treated with the same estradiol implant, along with subcutaneous implants (11 cm × 0.48 cm) containing 10% progesterone w/w for 12 days. Daily transrectal ovarian ultrasonography and blood sampling was performed from 5 days before, to 9 days after the period of implantation. Blood samples were also taken every 12 min for a 6 h period on day −2, 6 and 13 prior to or after implant insertion (day 0, day of implant insertion). Pulsatility in the serum LH levels was eliminated by the implants ( P < 0.05). During the implantation period, the serum FSH peak amplitude was lower in ewes treated with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol ( P < 0.05). No follicular waves emerged during implant treatment in both groups ( P < 0.05) and the number of serum FSH peaks did not differ during implantation, compared to before implantation. During the implantation period, the number of small follicles did not differ in ewes with implants releasing estradiol and progesterone, compared to ewes treated with implants releasing only estradiol. To conclude, supra-physiological concentrations of estradiol completely eliminated the serum LH pulsatality and suppressed the follicular wave emergence, while the FSH secretory peaks that preceded the follicular waves were not affected. Supra-physiological concentrations of estradiol-17β with physiological concentrations of progesterone decreased the serum FSH peak amplitude, eliminated the serum LH pulses, but did not decrease the size of the small follicle pool in anestrous ewes.

Methoxychlor Directly Affects Ovarian Antral Follicle Growth and Atresia through Bcl-2- and Bax-Mediated Pathways

Methoxychlor (MXC) is an organochlorine pesticide and reproductive toxicant. While in vivo studies indicate that MXC exposure increases antral follicle atresia, in part by altering apoptotic regulators (Bcl-2 and Bax), they do not distinguish whether MXC does so via direct or indirect mechanisms. Therefore, we utilized an in vitro follicle culture system to test the hypothesis that MXC is directly toxic to antral follicles, and that overexpression of anti-apoptotic Bcl-2, or deletion of pro-apoptotic Bax, protects antral follicles from MXC-induced toxicity. Antral follicles were isolated from wild-type (WT), Bcl-2 overexpressing (Bcl-2 OE), or Bax deficient (BaxKO) mice, and exposed to dimethylsulfoxide (control) or MXC (1-100 microg/ml) for 96 h. Follicle diameters were measured every 24 h to assess growth. After 96 h, follicles were histologically evaluated for atresia or collected for quantitative PCR analysis of Bcl-2 and Bax mRNA levels. MXC (10-100 microg/ml) significantly inhibited antral follicle growth at 72 and 96 h, and increased atresia (100 microg/ml) compared to controls at 96 h. Furthermore, MXC increased Bax mRNA levels between 48-96 h and decreased Bcl-2 mRNA levels at 96 h. While MXC inhibited growth of WT antral follicles beginning at 72 h, it did not inhibit growth of Bcl-2 OE or BaxKO follicles until 96 h. MXC also increased atresia of small and large WT and BaxKO antral follicles over controls, but it did not increase atresia of large Bcl-2 OE antral follicles over controls. These data suggest that MXC directly inhibits follicle growth partly by Bcl-2 and Bax pathways, and increases atresia partly through Bcl-2 pathways.

The temporal relationship between patterns of LH and FSH secretion, and development of ovulatory-sized follicles during the mid- to late-luteal phase of sheep

The aim of the present study was to investigate the temporal relationship between the secretory pattern of serum LH and FSH concentrations and waves of ovarian antral follicles during the luteal phase of the estrous cycle in sheep. The growth pattern of ovarian antral follicles and CL were monitored by transrectal ultrasonography and gonadotropin concentrations were measured in blood samples collected every 12 min for 6 h/d from 7 to 14 d after ovulation. There were two follicular waves (penultimate and final waves of the cycle) emerging and growing during the period of intensive blood sampling. Mean and basal LH concentrations and LH pulse frequency increased ( P < 0.001) with decreasing progesterone concentration at the end of the cycle. Mean and basal FSH concentrations reached a peak ( P < 0.01) on the day of follicular wave emergence before declining to a nadir by 2 d after emergence. None of the parameters of pulsatile LH secretion varied significantly with either the emergence of the final follicular wave or with the end of the growth phase of the largest follicle of the penultimate wave of the cycle. However, mean and basal LH concentrations did increase ( P < 0.05) after the end of the growth phase of the largest follicle of the final follicular wave of the cycle. Furthermore, the end of the growth phase of the largest follicle of the final wave coincided with functional luteolysis. In summary, there was no abrupt or short-term change in pulsatile LH secretion in association with the emergence or growth of the largest follicle of a wave. We concluded that the emergence and growth of ovarian antral follicles in follicular waves do not require changes in LH secretion, but may involve changes in sensitivity of ovarian follicles to serum LH concentrations.

Antral follicle growth and endocrine changes in prepubertal cattle, sheep and goats

In the growing heifer calve, there is an early post-natal, gonadotrophin driven increase in ovarian antral follicle growth. The endocrine regulation of and reason for this initial stimulation of ovarian follicular development are not fully understood. This initial endocrine activity appears to be later held in check by negative feedback suppression mechanisms until the heifer is of a sufficient body size to initiate oestrous cycles and to reproduce. There is increasing evidence from recent ultrasonographic studies, performed in the same groups of prepubertal heifer calves, that the development of ovarian antral follicles and tubular genitalia occur in parallel. There appear to be two distinct periods of enhanced development of the reproductive organs, from 2 to 14 weeks of age and again from 34 to 60 weeks of age, or just prior to puberty. First ovulation in heifers is preceded by a gradual increase in pulsed LH secretion, which results in enhanced antral follicle development and oestrogen production. It was demonstrated that prepubertal heifers produced recurrent antral follicular waves; maximum sizes and life span of the dominant follicles of waves, as well as periodicity and FSH dependency of wave emergence were similar to those in adult cattle. In does, no Graafian follicles are seen at birth and total follicle numbers increase to 2 months of age, and then decline to 5 months of age. In ewe lambs, studies using transrectal ovarian ultrasonography showed that antral follicle recruitment and growth increased after the first 2 months of age and just before puberty. This bi-phasic pattern of changes in ovarian follicle recruitment and growth is strikingly similar to that in heifer calves, but it contrasts with earlier post-mortem examinations of ovaries in ewe lambs. Unlike in cattle and adult ewes, the rhythmic pattern of follicular wave emergence was not established in pre- and peripubertal ewe lambs. The early increase in antral follicle numbers and size in ewe lambs may be, at least in part, due to changes in FSH release and potency, and enhanced follicle production prior to first ovulation is probably caused by an increase in the frequency of LH pulses.

Ultrasonographic study of the effects of the corpus luteum on antral follicular development in unilaterally ovulating western white-faced ewes

The objective of this study was to examine the local effects of the corpus luteum (CL) on ovarian antral follicle development by looking at follicle populations and dynamics in ovaries with or without CL, in unilaterally ovulating ewes, using a retrospective analysis of daily ultrasonographic records. The present report summarises the data from the first luteal phase of the breeding season (August–October; n=4), a luteal phase in the mid-breeding season (November–December; n=5), the last luteal phase of the breeding season (January–March; n=5), and the luteal phase after GnRH-induced ovulations in mid-anoestrus (May–June; n=4) of western white-faced ewes. Mean daily numbers of 3 mm follicles that did not grow any larger were significantly reduced in the CL-containing ovaries of ewes at all periods of study except for the transition to anoestrus. With all scanning periods combined, daily numbers of 3 mm follicles not growing further increased ( P<0.05) between day 6 and 15 after ovulation in the CL-containing ovaries. Based on mean data for the whole periods of observation, the non-CL-bearing ovaries of ewes in the transition to anoestrus had fewer ( P<0.05) follicles growing from 3 to ≥5 mm in size before regression compared with the mid-breeding season and mid-anoestrus. The lifespan of follicles reaching ≥5 mm in diameter was shorter ( P<0.05) in the CL- compared with non-CL-containing ovaries of anoestrous ewes induced to ovulate with GnRH ((6.5±1.3) and (9.0±1.0) days, respectively). Circulating concentrations of progesterone were lower during both transitional periods (into and out of anoestrus) and mid-anoestrus than during the mid-breeding season ( P<0.001), and were less during anoestrus than during both transitional periods ( P<0.05). It was concluded that CL/luteal structures locally suppressed the growth of ovarian antral follicles to the 3 mm size-range except during the transition to anoestrus, but that there was no inhibitory effect of the CL on the growth of ovarian follicles to larger diameters. The presence of CL/luteal structures did not affect the length of the lifespan of follicles reaching ≥5 mm in diameter nor the number of ovulations per ovary in cyclic ewes, but shortened large follicle lifespan in anoestrous ewes. Variations in peripheral concentrations of progesterone across the breeding season and between the breeding season and anoestrus did not alter the lifespan of large antral follicles. In the transition to anoestrus and during mid-anoestrus, the presence of the CL in an ovary appeared to maintain follicle development to ovulatory sizes and to increase the rate of turnover of large antral follicles, respectively.

Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes.

Daily transrectal ultrasonography of ovaries was done in seven Finn ewes during three 17-day periods from May to July. Blood samples were collected each day for estimation of the serum follicle-stimulating hormone (FSH), oestradiol and progesterone concentrations, and also every 15 min for 6 h, halfway through each period of ultrasonographic examination, to determine the patterns of gonadotropic hormone secretion. Four ewes ceased cycling from March to mid-April (ewes entering anoestrus early) and three in May (ewes entering anoestrus late). In all ewes cyclicity resumed during the period from mid-August to mid-September. The growth of ovarian antral follicles to periovulatory sizes of >/=5 mm in diameter was seen at all stages of anoestrus. An average of four waves of follicular development (follicles growing from 3 to >/=5 mm in diameter before regression) with a periodicity of 4 days were recorded during each of the three scanning periods. There was a close temporal relationship between days of follicular wave emergence and peaks of successive FSH fluctuations. Ewes entering anoestrus late exceeded ewes that became anoestrus early in numbers of large (>/=5 mm in diameter) ovarian antral follicles and maximum follicle diameter. Peak concentrations of transient FSH increases were higher (P<0.05) in ewes entering anoestrus late than in ewes entering anoestrus early. The secretion of luteinising hormone, (LH; mean and basal level, and LH pulse frequency, but not amplitude) was lowest during the month of June in all ewes. Oestradiol production was markedly suppressed throughout anoestrus. Peaks of progesterone secretion appeared to occur at regular intervals and were associated with the end of the growth phase of the largest follicles of sequential waves. In conclusion, the growth of ovarian follicles to ostensibly ovulatory diameters is maintained throughout anoestrus in Finn ewes and periodic emergence of follicular waves is correlated with an endogenous rhythm of FSH secretion. The present study also provides evidence for the inverse relationship between the time of the onset of seasonal anoestrus and the number and size of antral follicles developing throughout anoestrus in Finn ewes, and indicates that differences exist in both the secretion of and ovarian responsiveness to gonadotropic hormones among early and late anoestrous ewes.

Insulin-like growth factor-II mediates the steroidogenic and growth promoting actions of follicle stimulating hormone on human ovarian pre-antral follicles cultured in vitro.

FSH is important for ovarian antral follicle growth and steroidogenesis, processes in the human that are believed to be mediated by IGF-II. The objective of this study was to determine if human ovarian pre-antral follicles are also FSH- and IGF-II-responsive, since the clinical questions and mechanisms underlying the effects on the pre-antral follicle pool of exogenously administered gonadotropins for fertility therapy and elevated endogenous gonadotropins in the perimenopause remain unanswered. Class 2 preantral follicles were isolated from human premenopausal ovaries (n=6) and cultured in vitro with androstenedione and either no additives or with FSH or IGF-II. FSH (100 ng/mL) stimulated estradiol (E2) production by 3.58 +/- 0.4 fold over 48 hr, compared to controls without FSH. This effect was completely inhibited in the presence of the IGF-II antagonist, IGF binding protein-4 (IGFBP-4). IGF-II also stimulated E2 production by preantral follicles with doses as low as 1 ng/mL and within 24 hr of treatment. Maximal response of 3- to 9-fold above control was achieved with 100 ng/mL of IGF-II between 96-120 hr of culture. IGFBP-4 completely inhibited E2 production to basal levels. FSH stimulated IGF-II mRNA in pre-antral follicles about 4-fold, determined by RT-PCR. FSH also stimulated follicle growth, determined by light microscopy, 50-68% over 48 hr, compared to controls (P<0.001), a process that was inhibited in the presence of IGFBP-4. Cumulatively, these data support IGF-II as a mediator of FSH action on human preantral follicles.

Ovarian follicular dynamics during anoestrus in ewes.

The aim of the present study was to document ovarian antral follicle dynamics throughout seasonal anoestrus in sheep. Daily transrectal ultrasonography was performed during four 17 day scanning periods from March to July in Western White-faced crossbred ewes. Blood samples were collected each day with ultrasonographic scanning for measurement of serum concentrations of FSH, oestradiol and progesterone. Blood samples were also taken every 15 min for 6 h, mid-way through each period of ultrasonographic examination, to determine the patterns of secretion of gonadotrophic hormones. Hormonal data were then related to observed changes in follicular populations and the patterns of antral ovarian follicle turnover. Ultrasonography showed that the ovaries of anoestrous ewes remained active and that the largest ovarian antral follicles grew to a periovulatory size (> or = 5 mm in diameter) at all stages of anoestrus. The total number of all ovarian follicles > or = 3 mm in diameter was lower during early anoestrus compared with at mid-anoestrus because of a significantly smaller number of small (3 mm) and medium (4 mm) ovarian follicles. The largest ovarian follicles (attaining > or = 5 mm in diameter before regression) exhibited a wave-like pattern of growth; an average of three waves of follicular development were recorded in sheep during each of the four 17 day scanning periods in anoestrus, with follicular waves emerging approximately every 5 days. This rhythmic pattern of follicular emergence was found to be associated with the occurrence of fluctuations in serum FSH concentrations. The growth rate of the largest follicles of the wave increased significantly from early to late anoestrus in sheep. In addition, ovarian follicles not growing beyond 3 mm in diameter showed organized patterns of growth and regression; their numbers tended to be lower (P = 0.09) at 3 days before and on the day of follicular wave emergence. Some ewes were seen to maintain synthesis of progesterone throughout anoestrus. This submaximal progesterone secretion tended to occur at irregular intervals and was not coupled with changes in concentrations or patterns of gonadotrophin release, ovulations or detectable morphological luteinization of ovarian antral follicles. It was concluded that the growth of ovarian antral follicles to an ovulatory size was maintained throughout anoestrus in ewes, with a transient shift in the number of small and medium-sized follicles during mid-anoestrus, and that the periodic emergence of waves of large follicles (> or = 5 mm in diameter) occurred in synchrony with an endogenous rhythm of FSH secretion.

Abnormal growth of ovarian antral follicles in breast cancer patients

Ovarian antral follicles from patients with breast cancer were compared with follicles from healthy women. Steroid levels in the follicular fluid and the health status of the follicles were evaluated. Follicles were judged to be healthy or atretic by flow cytometric determinations of the deoxyribonucleic acid content of aspirated granulosa cell nuclei. Fifteen of the 25 follicles (60%) from the cancer patients contained unmeasurable or abnormally low steriod levels (i.e., less than 100 ng/ml) which were significantly (P less than 0.001) lower than in follicles of the same health status from healthy women (500 to 1000 ng/ml). It is speculated whether substances other than the usual follicular steriods are produced by the cancer patients, which stimulate mitotic activity of the granulosa cells.