Effects of luteinizing hormone treatment on oogenesis in ovarian germ cells of the chick (Gallus domesticus)

The effect of the luteinizing hormone (LH) on the oogenesis of ovaries from newly-hatched chicks treated in vivo on days 13, 15, and 17 of embryonic development was analyzed. Changes in oogonial proliferation, meiotic prophase, degeneration of germ cells, and primordial follicular organization were determined. Results indicate that the total number of germ cells was not affected by the LH treatment, but significant differences existed in the number of oogonia and oocytes between the ovaries of control and LH-treated chicks. LH treatment increased the percentage of oocytes and diminished the percentage of oogonia. The mitotic activity of oogonia and degeneration of germ cells decreased, but the number of follicles during development increased in LH-treated ovaries. These findings suggest that LH treatment might trigger a cascade of endocrine events, resulting in inhibition of oogonial proliferation and induction of the meiotic prophase and follicle formation.     

Oestradiol Induced Inhibition of Neuroendocrine Marker Expression in Leydig Cells of Adult Rats

The objectives of this work were to determine the changes in the expression of neuroendocrine markers in Leydig cell by oestradiol treatment, and to determine whether testosterone is able to recover partially the effects of hormonal suppression induced by oestradiol. Adult male rats were injected daily with either 50 microg of oestradiol or oestradiol plus testosterone propionate (25 mg every 3 days) for 15 days. The animals were sacrificed and testicles were dissected and processed by routine histological protocols. FSH and LH serum levels were determined by radioimmunoassay. The visualization of antigens was achieved by the streptavidin-peroxidase immunohistochemical method. Antibodies against chromogranin A (CrA), S-100 protein (S-100), P substance (PS), synaptofisin (SYN), neurofilament protein (NF), gliofibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were used. The mean LH and FSH serum concentrations were consistently suppressed with hormonal treatments. Intermediate filaments (NF and GFAP) showed no difference in their expression. The expression of S-100, NSE and SYN was significantly lower in both hormone-treated groups. In oestradiol-treated rats, the immunoreactivity of CrA and SP decreased significantly but was restored after testosterone supplementation. Although the nature and functions of many of these substances in Leydig cells remain unknown, these results are consistent with the hypothesis that the expression of some neuroendocrine markers is hormonally controlled.     

Effect of luteinizing hormone on the right regressing ovary of newly hatched chicks treated during embryonic development

We studied the histologic and stereological changes induced in the right ovary of newly hatched chicks treated with LH during their embryonic development. Results indicate that LH administration causes a diminution in size and total volume (P < 0.01) of the right ovary, as well as a decrease in the total volume of lacunar channels, blood vessels, and interstitium. Other changes obtained after LH treatment were a reduction (P < 0.001) in the number of germ cells, as well as an increase in the total volume of interstitial cell cords (P < 0.01). This expansion is due to the increase of cellular volume of interstitial cells (P < 0.001) and not to their number, which decrease in the LH-treated right ovary. All these modifications were similar to those occurring in the regressing right ovary during development. The findings suggest that the right ovary of the newly hatched chick is able to respond to LH treatment during embryonic development, inducing marked histologic changes that accelerate its regression.     

Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells Cultured In Vitro Under Serum‐Free Conditions

The objective of this study was to examine the effects of FSH and LH on oestradiol‐17β and progesterone production by buffalo granulosa cells cultured under serum‐free conditions. Granulosa cells (3 × 10⁵) from small (≤5 mm diameter) follicles were cultured for up to 4 days in 48‐well plates coated with 3.3 μg/cm² fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F‐12 Ham (1 : 1 ratio) supplemented with 10^?7 m androstenedione, 5 μg/ml human apo‐transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol‐17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol‐17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol‐17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum‐free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.     

Production of immunoactive inhibin by bovine granulosa cells in serum-free culture: Effects of exogenous steroids and FSH

Granulosa cells from pooled bovine follicles were cultured under chemically-defined (serum-free) conditions to study the effects of exogenous steroids and FSH on production of immunoactive (ia) inhibin, oestradiol and progesterone. Levels of ia-inhibin in media samples and cell lysates were measured by radioimmunoassay (RIA) using an antiserum raised against a synthetic fragment of human inhibin -subunit [hI(1–32)].

Cells secreted measurable amounts of ia-inhibin, oestradiol and progesterone for at least 7 d of culture, although intracellular levels of inhibin were very low, indicating that newly-synthesized ia-inhibin is rapidly released from the cells. Treatment with androstenedione (0.2μmol/l) or testosterone (0.2μmol/l) increased ia-inhibin secretion markedly; levels on Day 5 of culture were approximately 6-fold (P<0.005) higher than control values. In contrast, treatment with the non-aromatizable androgen dihydrotestosterone (DHT; 0.2μmol/l) resulted in only a one- to two-fold increase (P<0.05) over control values (Day 5). Addition of exogenous oestradiol (8nmol/l) markedly increased ia-inhibin secretion (8–9 fold on Day 5; P<0.05) compared with basal levels, whereas progesterone had no effect. Secretion of oestradiol, undetectable in the absence of exogenous androgens, rose daily in the presence of either androstenedione or testosterone, levels rising approximately 6-fold and 9-fold respectively over a 4-d treatment period. Progesterone secretion increased 2-fold over the culture period and was unaffected by any steroid treatment.

Treatment with ovine FSH (10ng/ml) alone stimulated secretion of progesterone over basal levels (3-fold higher on Day 6; P<0.005), but did not affect output of either ia-inhibin or oestradiol. However, exposure to FSH in the presence of androstenedione not only promoted a further 4-fold increase in progesterone output but also led to a dose-dependent suppression of both ia-inhibin (90% lower on Day 6; P<0.001) and oestradiol (80% lower on Day 6; P<0.001) secretion compared to cells treated with androstenedione alone.

These observations indicate that the secretion of ia-inhibin by bovine granulosa cells in culture is positively regulated by oestradiol, implying an autocrine/paracrine role for this hormone in control of ovarian inhibin production. The ability of aromatizable androgens to stimulate secretion of inhibin, coupled with the inability of the non-aromatizable androgen DHT to elicit such an effect, suggests that inhibin output is largely unaffected by androgens prior to their conversion to oestradiol. The absence of any change in output of ia-inhibin or oestradiol following treatment with exogenous progesterone argues against a local role for this steroid hormone in regulation of inhibin or oestradiol production in the bovine follicle. Finally, the observation that co-treatment with FSH and andostenedione not only stimulated progesterone output but also suppressed secretion of ia-inhibin and oestradiol, indicates a synergistic positive effect of FSH and androgens on cellular luteinization.     

猪卵巢发育的组织学特点及糖组织化学

运用组织学常规石蜡切片-HE染色和过碘酸-雪夫染色（PAs）方法,分别对3日龄及165日龄育成猪卵巢组织结构、各级卵泡的发育变化特点及其黏多糖分布情况进行了研究。结果显示,3日龄的猪卵巢皮质、髓质界限模糊,皮质部分由外向内依次分布着密集的卵原细胞和卵原细胞巢、共质体样的卵原细胞群,皮质深层与髓质相邻处分布着合胞体样的卵母细胞簇状结构和卵泡。育成猪卵巢中,皮、髓质界限明显,能观察到原始卵泡、初级卵泡、次级卵泡及近成熟卵泡,但是很少能见到黄体的结构。PAS染色结果表明,3日龄的猪卵巢中PAS阳性反应主要分布于卵原细胞周围的基质、卵原细胞巢和合胞体的外膜上,以及原始卵泡周围的基质、初级卵泡的卵泡膜及刚形成的透明带上,此外卵巢的白膜、髓质的结缔组织、血管壁及卵巢网周围也可见到广泛的阳性着色。育成猪卵巢中,PAS阳性反应主要分布于基质、生长卵泡的卵泡膜、卵泡的透明带、次级卵泡的卵泡液、黄体被膜及髓质的结缔组织和血管壁。     

Ultrastructural Aspects of the Prenatal Bovine Ovary Differentiation with a Special Focus on the Interstitial Cells

The aim of this investigation was to study the ultrastructural features during the development of fetal bovine ovaries (crown rump length ranging from 11.4 to 94.0 cm). An interesting observation was the occurrence of big elongated cells containing a variety of electron dense granules and light homogenous vacuoles/bodies. They were located between the stroma cells surrounding the germ cell cord ends, adjacent to the first formed primordial follicles, typically situated near blood vessels. ER alpha and ER beta receptor positive cells could be detected in the same regions by means of immunohistochemistry. Intercellular bridges linked the germ cells nests oogonia. Germ cell cords consisted of centrally located, large, pale oogonia, surrounded by elongated somatic cells with very long cytoplasm extensions. Primordial follicles with flat pale follicular cells could be observed on the inner end of the cords. Extrusions of the outer nuclear membrane could often been recognised in voluminous oocytes.     

Cellular changes in the anterior pituitary of the domestic fowl during growth, sexual maturity and laying

All the cell types in the hen's anterior pituitary varied in number during the period from 10 weeks to adulthood; there was often an associated change in apparent functional activity. The cells most affected were the gonadotrophs (both FSH and LH) and luteotrophs; changes in these cells were associated with the onset and maintenance of sexual maturity. Follicle‐stimulating hormone cells were the first to increase in number and activity and this was correlated with a general increase in steroid output from the ovary, as measured by the oviduct weight increase. With the onset of follicular maturation LH cells became obvious. Prolactin cells increased in number with the presumed general increase in circulating oestrogen. Acidophilic somatotrophs decreased with increasing age; thyrotrophs became more numerous about sexual maturity and it is thought that this reflects the general increase in metabolic rate which occurs at this time.     

不同月龄绵羔羊卵泡发育的比较研究

本研究旨在比较1、3月龄绵羔羊激素处理后卵巢、子宫及血液中促卵泡激素(FSH)变化,研究不同月龄对羔羊卵泡发育的影响。通过对1、3月龄羔羊进行FSH和孕马血清促性腺激素(PMSG)处理,比较羔羊在激素处理前后血液中FSH水平和卵巢、子宫大小变化,卵巢上2～8 mm卵泡的数量和卵母细胞体外成熟、受精后胚胎的发育情况。结果表明:1月龄羔羊实验组体内整体FSH水平高于3月龄羔羊(P<0.05)。1月龄羔羊注射外源激素后两侧卵巢可获卵母细胞数(42.3±2.5、36.8±1.1)枚及体外受精囊胚发育率(16.33%±0.96%)显著高于3月龄羔羊卵母细胞数(10.0±0.7、8.5±0.6)枚及囊胚率(9.29%±1.55%)(P<0.05)。羔羊进行超数排卵处理时卵巢上卵泡发育与血液中FSH水平密切相关,且较高的FSH水平预示着较多的卵泡发育。     

In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5–0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.     

Follicle stimulating hormone increases serum oestradiol-17 beta concentrations, number of growing follicles and yolk deposition in aging hens (Gallus gallus domesticus) with decreased egg production.

1. The aims of this study were to determine if the number of small yellow follicles (SYF) and large white follicles (LWF) in ovaries of young and old hens differed; and if injection of old hens with follicle stimulating hormone (FSH) changed growth of and yolk deposition into follicles of old hens. 2. Ovaries were removed and follicles were divided according to size and condition and counted. The number of normal SYF and LWF was decreased in old hens compared to young hens, whereas the number of atretic follicles was greater in old hens compared to young hens. 3. Old hens were injected subcutaneously with saline containing 0.1% bovine serum albumin (BSA, vehicle) or 12.5, 50, 200, 400 micrograms porcine (p) FSH or 25 or 50 IU pregnant mare's serum gonadotropin (PMSG) for 5 consecutive days. Blood samples were taken on days 1 and 5 before FSH and PMSG injection and 2 h after FSH and PMSG injection on day 5. Sudan black and Sudan red dyes were injected intravenously on alternative days to monitor yolk deposition into follicles of the hierarchy removed after the fifth day of FSH treatment. 4. Treatment with 200 micrograms pFSH or 25 IU PMSG for 5 d increased serum progesterone (P4) concentrations as compared to controls. Injection of hens with FSH caused a linear dose dependent increase in serum oestradiol-17 beta (E2) concentrations, dose dependent increase in SYF and LWF, and dose related decrease in number of atretic SYF and LWF. The hierarchy of the ovary was disrupted with PMSG, but not FSH. Larger doses of FSH increased the number of small follicles (10 mm diameter) and yolk deposition. 5. We conclude that small follicles which have not entered the rapid growth phase are responsive to FSH. The increased yolk deposition following FSH treatment may have been a direct effect of FSH or may have been caused by the elevation of serum E2 concentrations in response to FSH treatment. It is possible that old chickens may produce inadequate amounts of FSH which result in decreased rate of follicular growth and ultimately decreased egg production.     

Ovarian stimulation and ultrasound-guided oocyte retrieval in baboon (Papio Cynocephalus Anubis) during pituitary suppression with a GnRH agonist

The objective of this study was to investigate whether baboon females respond to an ovarian stimulation protocol incorporating pituitary suppression with a GnRH agonist (GnRHa) and either highly purified human FSH (hphFSH) or recombinant human FSH (rhFSH) with follicular development and oocyte maturation. A modified human ovulation induction protocol was applied to 5 adult female baboons with a history of regular menstrual cycles (33-34 days). A long-acting GnRHa implant containing goserelin acetate was placed subcutaneously (s.c.) on Days 22-24 of their menstrual cycle. Concentrations of serum oestradiol (E2), progesterone (P4) and human FSH were obtained by ELISA. Menses occurred approximately 10 days after GnRHa implantation. Daily hphFSH or rhFSH (75 IU i.m.) treatments were started approximately 10 days following menses. When the majority of follicles were > or = 5 mm in diameter and the E2 levels had reached a maximum, hCG (2000 IU i.m.) was administered to induce final maturation of oocytes and ovulation. Thirty to 34 h after hCG administration, transabdominal follicular aspiration was performed using a variable frequency transvaginal transducer with ultrasound. A total of 71 oocytes were collected from 4 animals (average: 17). The meiotic maturity of oocytes was evaluated 3 h after retrieval. Ninety-one percent of oocytes were in metaphase 2 and of grades I and II which are appropriate for in vitro insemination.     

Effect of luteinising-hormone in vivo on ovarian cell subpopulations of newly-hatched chicks

1. We analysed the number and size of different ovarian cell subpopulations of newly-hatched chicks by ovarian cell suspension count and morphometric/stereological methods as well as delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta HSD) activity in these cells treated in vivo with LH during embryonic development. 2. Fertile White Leghorn eggs received 1 microg LH applied to the chorioallantoic membrane on days 13, 15, and 17 of incubation. All animals were killed within 24 h after hatching, the left ovary was dissected and processed. 3. The results indicate that the number of germ, pregranulosa, interstitial and undifferentiated cells was not affected by LH treatment. However, we observed an increase in the size of individual interstitial cells of the ovarian medulla. In these cells, delta5-3beta HSD activity was increased in response to LH. 4. These findings suggest that LH does not exert a proliferative effect on the cells of the prefollicular ovary of the chick and that interstitial cells can be target cells for LH, increasing their steroidogenic activity due to LH treatment.     

Reproductive characteristics of stallions during the breeding and non-breeding season in a tropical region

The objective of this study was to investigate reproductive characteristics of stallions at a tropical zone in the breeding and non-breeding seasons. The following parameters were assessed: testicular volume; semen quality; and serum concentrations of LH, FSH, and testosterone; in addition to the percentages of germ cells and proportions of germ cells/Sertoli cells by testicular cytology in stallions. Semen was collected from eight adult stallions twice a week during a 12-week period in both seasons (6?weeks before and 6?weeks after the summer and winter solstices). Jugular blood samples were collected periodically for hormone analysis by radioimmunoassay during the same periods. Testicular measures and cytological samples were taken at the end of each period. Mean concentration of testosterone was significantly higher (P?=?0.04) during the breeding season and the proportion of Sertoli cells/100 germ cells in cytological smears was significantly lower during the breeding season (P?=?0.0001). Effects of season were not significant either for testicular volume or for any semen parameter (P?>?0.05). Seasonal changes in the mean concentrations of LH and FSH were not observed (P?>?0.05). There were also no significant differences in the mean percentages of germ cell types between both seasons (P?>?0.05). Lack of seasonal differences in the testicular volume and semen parameters of tropical stallions are probably due to the small variation in duration of natural light between the observed periods, slightly under 3?h.     

Apoptosis and proliferation during seasonal testis regression in the brown hare (Lepus europaeus L.)

Testes samples of 52 brown hares (Lepus europaeus L.), sacrificed between July and January, were subjected to immuno histochemical analysis. The terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method was applied to detect apoptosis; and antibodies to proliferating cell nuclear antigen (PCNA) were used to evaluate cell proliferation in the testes. In the seminiferous epithelium, the apoptotic processes were evident from August to early November with maximal values in September. Cell death in germ cells occurs predominantly during the prophase of the first meiotic division. In July, and from mid-November onwards, only the occasional TUNEL-positive cells can be seen. The proliferation of germ cells continues during the testis regression phase. The average number of PCNA-positive cells decreases slightly from September onwards and rises again in mid-November.     

In Vitro Effect of the Reproductive Hormones on the Oxidative Burst Activity of Polymorphonuclear Leucocytes from Cows: A Flow Cytometric Study

In this study, the effect of reproductive hormones and substances with hormonal activity on the oxidative burst activity of blood polymorphonuclear leucocytes (PMN) high yielding dairy cows was evaluated. Different concentrations of: progesterone, oestradiol 17β, FSH, LH, GnRH, cortisol and PGF2α were incubated in vitro for 4 h with PMN of seven high milk yielding cows, during the period of anoestrous postpartum. Controls were run in parallel in which each hormone was replaced by its solvent. After incubation with hormones the competence of PMN to generate H₂O₂ was monitored by flow cytometry. A down‐regulation on the oxidative burst activity of PMA‐stimulated PMN was observed when cells were incubated with progesterone. Significant (p ≤ 0.001) differences between control and progesterone incubated cells were observed from 6.56 μg/ml. The same predisposition was observed when PMNs were incubated with cortisol. Besides for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Concerning oestradiol 17β, an increase on H₂O₂‐production was observed when PMN were incubated with 15 pg/ml and 45 pg/ml of this steroid (p ≤ 0.05), followed by a depression of the cell’s activity when unphysiological concentrations were employed. Significant (p ≤ 0.05) differences between the obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol. No statistical differences were observed in the metabolic burst activity of PMN incubated with FSH, GnRH and LH when compared with the results obtained by the control.     

Thirteen-weeks subcutaneous treatment with high dose of natural sex hormones in rats with special reference to their effect on the pituitary-gonadal axis. I. Oestradiol

A high dose of oestradiol (0.3 mg/kg/day) was administrated subcutaneously to male and female rats daily for 13 weeks. The effects of hormonal treatment on various parameters were studied. The results revealed that treatment with oestradiol resulted in alopecia, retarded hair regrowth, decreased body weight and food consumption and reduced Hb, PCV and total RBCs. Neutrophilic leucocytosis, elevated ESR, and decreased blood glucose levels were also observed. Atrophy of the ovaries, testes and secondary sex organs was also recorded. The uterus of the oestradiol treated rats displayed endometrial epithelial cell hyperplasia and hypertrophy of the myometrium. The pituitary gland of the rats with oestradiol had a significant increase in the number of PRL and ACTH cells together with cytological criteria indicative of increased secretory activity; the gonadotropin-producing cells showed involutionary changes. The mammary glands of the oestradiol treated rats showed maximal stromal and ductal proliferation and minimal acinar proliferation.     

The impact of bisphenol S on bovine granulosa and theca cells

Bisphenol S (BPS) is an endocrine‐disrupting chemical with multiple potential mechanisms of action, including as an oestrogen receptor agonist. BPS is increasingly used in plastics and thermal receipts as a substitute for bisphenol A, which has been phased out due to concerns about human health implications. The ability of BPS to alter female reproductive function in mammals has not been widely studied, despite the importance of normal hormone signalling for female reproduction. The aim of this study was to investigate how BPS (in a wide range of doses, including very low doses) affects granulosa cell and theca cell steroid hormone production and cell viability in the bovine. Granulosa cell oestradiol production was stimulated when cells were exposed to 100 μM BPS under basal conditions, but there was no effect of BPS when cells were stimulated with follicle‐stimulating hormone (FSH). Additionally, there was no effect of BPS on granulosa cell progesterone production or cell viability under basal or FSH‐stimulated conditions. BPS did not affect theca cell androstenedione or progesterone production, or theca cell viability under basal or luteinizing hormone‐stimulated conditions. This study suggests for the first time that BPS may alter oestradiol production by bovine granulosa cells, albeit at a concentration that is unlikely to be physiologically relevant. Further studies are needed to determine the effects of BPS on the bovine oocyte and on other functions of follicular cells.     

Effect of oestradiol on mast cell number and histamine level in the mammary glands of rat

Variations of mast cell number, histamine concentration and oestrogen receptor (ER) expression in mammary glands with the fluctuation of plasma oestradiol level were identified either in the intact rats at different oestrous stages or in the ovary-ectomized rats administrated with different doses of oestradiol benzoate. The results showed that the number of mast cells and histamine concentration fluctuated concomitantly with plasma oestradiol level during the oestrous cycle. More mast cell number and higher histamine concentrations were observed in the oestrous stage than that in the prooestrous and dioestrous stages. Ovariectomy decreased the mast cell number and histamine concentration, which were reconstituted by exogenous oestradiol. ER was mainly found in the nuclear of epithelial cells and interstitial cells of mammary glands. In addition, ER was also expressed in the cytoplasm of some stromal cells. These stromal cells were verified to be mast cells. In conclusion, our results suggested that oestradiol modulated mast cell number and its degranulation in the mammary gland through the ERs pathway.     

The Modulation of Gonadotrophic Hormone Action on the Ovary by Paracrine and Autocrine Factors

It has been hypothesized that the physiological basis of follicle selection is the differential expression of factors, which modulate the action of gonadotrophins on follicular cells, at key points during the process of follicle development. The aim of this research was to test this hypothesis by identifying factors that can enhance or attenuate the action of the gonadotrophins in stimulating follicle development using both in vivo and in vitro models. Experiments in vivo utilized sheep with an ovarian autotransplant to allow intra-arterial infusion of putative local factors and exposure of the ovary to high local concentrations. Experiments in vitro utilized physiological serum-free cell culture systems for both granulosa and theca cells that allow gonadotrophin-induced differentiation in vitro. The putative local factors tested included insulin-like growth factor-I (IGF-I LR3 analogue), transforming growth factor alpha (TGF alpha) or epidermal growth factor (EGF) and inhibin A. IGF-I stimulated both cellular proliferation and hormone production by both granulosa and theca cells in vitro and similarly stimulated ovarian follicle development and ovarian androgen and oestradiol secretion in vivo. Both TGF alpha and EGF stimulated granulosa and thecal cell proliferation in vitro in a dose-responsive manner and concomitantly inhibited hormone production, whereas intra-arterial infusion of TGF alpha in vivo resulted in induction of atresia in large antral follicles and an acute fall in ovarian hormone secretion. Inhibin A in vitro augmented gonadotrophin stimulated androgen and oestradiol production by thecal and granulosa cells, respectively, but had no effect on cell number. Paradoxically, intra-arterial infusion of inhibin A resulted in an acute depression in ovarian steroid secretion. This depression, however, was also associated with an acute depression in circulating FSH concentrations. In conclusion, these data provide strong support for the hypothesis that factors can modulate the action of gonadotrophins on follicular cells to augment (IGF-I, inhibin A) or inhibit (TGF alpha/EGF) granulosa and thecal cell differentiation. The challenge for the future in this area of research is to understand how these factors interact to enable one follicle to be selected from an ovulatory cohort.