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Inhibin-A and estradiol content of sheep follicles and their association with follicular diameter
Small Ruminant Research 42 (2001) 179±183
Inhibin-A and estradiol content of sheep follicles and their association with follicular diameter F. Shidaifat* Department of Basic Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030 Irbid, Jordan Accepted 27 June 2001
Abstract This study was conducted to investigate dimeric inhibin-A and estradiol content of sheep antral follicles and their association with follicular size. Follicular content of inhibin-A and estradiol in small, medium and large antral follicles were determined by enzyme immunosorbent assay. The results showed that both estradiol and inhibin-A content of follicular ¯uid increased as the follicular size increased from small to medium and large-sized antral follicles. Although estradiol and inhibinA content were signi®cantly higher, in large antral follicles compared to small and medium-sized follicles, no signi®cant increase was observed between medium and small follicles. These results indicated that estrogenic active follicles are capable of producing parallel quantities of inhibin-A. To investigate a possible local role for inhibin-A on estrogenic capacity of sheep granulosa cells, cells obtained from antral follicles were cultured in a chemically de®ned (RPMI-1640) in the presence or absence of 100 ng/ml inhibin for 24, 48 and 72 h. The results showed that granulosa cells acquire an enhanced ability for estradiol production in the absence of inhibin as the time progresses. Inhibin treatment showed no effect on estradiol production of granulosa cells. As inhibin-A is associated with follicular size and their estradiol content, it exerts no effect on estrogenic capacity of granulosa cells. Probably, inhibin-A exerts its effect on sheep follicular development through endocrine and/or paracrine routes rather than an autocrine route. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Inhibin; Estradiol; Granulosa cells; Follicles
1. Introduction Follicular growth occurs as a series of waves throughout the reproductive cycle of sheep. The emergence of these waves have been shown to be associated with a transient increase in serum concentration of follicle stimulating hormone (FSH), suggesting a major role of FSH in the regulation of sheep follicular development (Bartlewski et al.,
* Tel.: 962-2-7095111; fax: 962-2-7095123. E-mail address: [email protected] (F. Shidaifat).
1999; Ginther et al., 1995; Souza et al., 1998). However, the ®ne tunings of FSH secretion by the pituitary gland and the subsequent monitoring of follicular development require a complex interaction with ovarian factors (Ying, 1988; Findlay, 1993). Of those, inhibin, a gonadal-derived glycoprotein is a heterodimer composed of an a-subunit linked by disul®de bonds to either of two bA or bB subunits to form active dimers known as inhibin-A and inhibin-B, respectively (Burger and Igarashi, 1988). Although, mRNA for a, bA and bB inhibin subunits were found to be expressed speci®cally by the granulosa cells of developing healthy follicles in sheep
0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 2 5 8 - 9
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F. Shidaifat / Small Ruminant Research 42 (2001) 179±183
(Braw-Tal, 1994; Tisdall et al., 1994; Campbell et al., 1999), circulating inhibin-A appears to be the predominant circulating form (Knight et al., 1998). The circulating pattern of inhibin-A and its relation to follicular development, has led to the suggestion that the largest dominant follicle is the main source of circulating inhibin-A (Souza et al., 1997, 1998). Although inhibin is characterized primarily by its ability to attenuate FSH secretion by the pituitary gland (Findlay et al., 1987; Rivier et al., 1991; Carroll et al., 1991), a local role in the regulation of ovarian function has been proposed (Campbell et al., 1995). Therefore, this study was conducted to determine the follicular content of inhibin-A and its association with estradiol and follicular size. In addition, a possible local effect of inhibin-A on granulosa cells differentiation was examined in vitro. 2. Materials and methods 2.1. Inhibin-A assay Follicular ¯uid was obtained from Awassi sheep ovaries, which were transported to the laboratory within 2 h post-mortem. The follicular ¯uid was completely aspirated from 24 individual follicles using a Hamilton syringe. The follicles were classi®ed as small (<3 mm), medium (3±6 mm) and largesized antral follicles (>6 mm). The granulosa cells were then removed by centrifugation for 5 min at approximately 200 g. Inhibin-A dimer concentration was determined by ultra sensitive two-site enzyme immunoassay (EIA) kit (SeroTec, Oxford, UK) according to the method of Groome et al. (1994). The follicular ¯uid was diluted in fetal calf serum and then pretreated with 6% sodium dodecyl sulfate and freshly prepared 6% hydrogen peroxide. The samples were then added to a microplate coated with monoclonal antibodies speci®c for bA subunit of inhibin and incubated overnight. At the end of this incubation period, the plate was then washed, and a second monoclonal antibody speci®c for the a subunit of inhibin was added. The secondary antibody is coupled to alkaline phosphatase. Inhibin dimer was detected by addition of alkaline phosphatase substrate, after the removal of the unreacted material by washing.
2.2. Estradiol 17b assay Follicular ¯uid content of estradiol 17b and that secreted by cultured sheep granulosa cells were evaluated by direct estradiol EIA kits (BioSource, Belgium). Absorbance was measured with Dynatech microplate reader at a wavelength of 450 nm. Awassi sheep ovaries, obtained from a local slaughterhouse, were transported to the laboratory within 2 h of slaughter. The ovaries were collected during September from sheep of 7±9 months old. After soaking in 70% ethanol for approximately 1 min, the ovaries were washed ®ve times with distilled water. Since granulosa cells from medium-sized follicles acquired enhanced aromatase activity and estradiol production, granulosa cells harvested from those follicles were used to evaluate the effect of inhibin-A on estradiol production. Granulosa cells were aspirated from antral follicles (3±5 mm in diameter) using a 20-gauge needle ®tted to a 10 ml syringe. The follicular ¯uid containing the granulosa cells was suspended in RPMI-1640 (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and antibiotic±antimycotic mixture (100 IU penicillin, 100 mg/ml streptomycin, and 250 mg/ml amphotericin) (GIBCO BRL, Grand Island, NY). Approximately 2:0 105 live cells in 1 ml RPMI1640, containing 10% FBS and antibiotic±antimycotic mixture, were initially seeded per well in 24-well culture plate (Costar, Corning, NY) and allowed to grow for 48 h in a humidi®ed incubator at 37 8C, 5% CO2 and 95% air. At the end of this culture period, cells reached about 90% con¯uency as assessed by the inverted microscope. The cells were then washed with RPMI-1640. To determine the effect of recombinant inhibin-A (Mayo et al., 1986) (R&D systems, Minneapolis, MN) on granulosa cells estradiol production, cells were cultured for additional 24, 48 and 72 h in the presence or absence of 100 ng/ml inhibinA. At the end of each treatment period, the media were collected for estradiol evaluation by EIA. Cell treatments were carried out in 1 ml of RPMI-1640 supplemented with 1 mg/ml insulin, 0.67 mg/ml transferrin and 0.55 mg/ml selenium (GIBCO BRL, Grand Island, NY). In addition, 10 mg/ml of low density lipoprotein (Sigma, St. Louis, MO) and 1 mM testosterone were used as substrate for steroid synthesis.
F. Shidaifat / Small Ruminant Research 42 (2001) 179±183
181
Least squares procedures were used to analyse inhibin-A and estradiol content using a model that included ®xed effect due to follicle size (small, medium and large). Signi®cant difference among treatments were based on Bonferroni multiple comparison test. 3. Results Sheep ovaries appeared to contain follicles of different sizes with only one large preovulatory follicle. The mean follicular ¯uid content of inhibin-A is shown in Fig. 1. The results showed a progressive increase in the follicular ¯uid content of inhibin-A as the follicle size increased. The mean follicular ¯uid content of inhibin-A was signi®cantly higher in largesized antral follicles compared to medium- and smallsized antral follicles. Although, inhibin content of medium-sized antral follicles was increased two-fold over that of small-sized antral follicles, no signi®cant difference was observed. This increase in the follicular content of inhibin-A of large antral follicles was associated with a progressive increase in estradiol content. The mean follicular ¯uid content of estradiol is shown in Fig. 2. Large size follicles contained higher concentration of estradiol (P < 0:01) compared to medium and small-sized follicles. Although estradiol content increased as the follicular size increased, the increase in estradiol content was signi®cant in large follicles only. The effect of inhibin-A on sheep granulosa cells estradiol production are presented for six replicate
Fig. 1. Pattern of sheep follicular content of inhibin-A in small, medium and large antral follicles. Inhibin-A follicular ¯uid content of eight individual follicles in each group was presented as the mean S:E:M. Bars with different letters are signi®cantly different.
Fig. 2. Pattern of sheep follicular content of estradiol in small, medium and large antral follicles. Estradiol follicular ¯uid content of eight individual follicles in each group was presented as the mean S:E:M. Bars with different letters are signi®cantly different.
Fig. 3. Time-effect of recombinant inhibin-A on basal estradiol production by cultured sheep granulosa cells. Data presented as the mean S:E:M of six replicate culture wells. Bars with different letters are signi®cantly different.
cultures of each treatment (Fig. 3). This experiment was repeated twice using different pools of granulosa cells harvested from medium-sized follicles. The time-effect of inhibin-A on estradiol production by cultured sheep granulosa cells has been presented. Cultured sheep granulosa cells acquired a progressive and signi®cant increase in their estradiol production on the absence of inhibin-A as the time progress. Inhibin-A treatment (100 ng/ml) of cultured sheep granulosa cells showed no effect on their estradiol production at the time interval tested. 4. Discussion The data presented here shed light on the microenvironment of the developing follicles in term of their
182
F. Shidaifat / Small Ruminant Research 42 (2001) 179±183
estradiol and inhibin content. Results of previous investigations of sheep ovarian follicular dynamics and its relation to circulating inhibin-A and estradiol provided indirect evidence that the granulosa cells of the dominant follicle are the main source of circulating inhibin-A and estradiol (Souza et al., 1997, 1998). The results of this study con®rm that proposal by showing a progressive increase in sheep follicular ¯uid content of dimeric inhibin-A and estradiol as the size of the follicles increased with peak values in the largest antral follicles. These results are consistent with the reported expression pattern of inhibin a, bA subunits (Braw-Tal, 1994, Tisdall et al., 1994; Campbell et al., 1999) and aromatase P450 (Huet et al., 1997; Tsonis et al., 1984) by sheep granulosa cells which have been shown to be stage-dependent and speci®c to granulosa cells of healthy antral follicles, but not to atretic follicles. The parallel changes in the pattern of follicular content of inhibin-A and estradiol re¯ected a close temporal relationship between the initiation and subsequent growth of the antral follicles through the various developmental stages, suggesting that the differentiated growing follicles are the main source of inhibin and estradiol. However, the results of this study indicated that medium and small-sized follicles, which present side by side at the same ovary also produced a substantial amount of both factors suggesting a possible contribution of these follicles to circulating inhibin and estradiol. The increased levels of both estradiol and inhibin-A have been shown to be conversely related to the circulating levels of FSH indicating a negative endocrine effect of inhibin-A on FSH secretion by the pituitary gland (Knight et al., 1998; Souza et al., 1997). However, interference with inhibin effect by immunization, which leads to an increase in the ovulation rate, cast doubts on the endocrine role of inhibin on FSH secretion, suggesting a local alternate pathway through which inhibin-A exerts its effect on follicular development (D'Alessandro et al., 1999; Campbell et al., 1995). Therefore, a possible local regulatory role of inhibin-A on follicular development was examined using granulosa cell culture. Granulosa cells of developing follicles acquired enhanced estrogenic activity as during sequential stages of differentiation. The results of this study provided evidence that the sheep granulosa cells of antral follicles are capable of synthesizing substantial quantities of estra-
diol suggesting a spontaneous differentiation of sheep granulosa cells in vitro. However, inhibin-A exerts no effect on the cultured granulosa cell estrogenic capacity. Since inhibin-A produced by differentiated granulosa cells exerts no effect on their differentiation, it appears that increased inhibin-A production is a result of granulosa cells differentiation. Although its autocrine effect on sheep granulosa cells differentiation seems unlikely, inhibin-A appears to have a paracrine effect on thecal androgen production (Hillier et al., 1991). Thecal androgen serves as an important precursor for estradiol production by granulosa cells which is required to support the high granulosa aromatization activities during the phases of follicular growth and differentiation. In short, the data presented here indicated a positive association of inhibin-A and estradiol content of follicular ¯uid with follicular size suggesting that those factors are markers of follicular development and differentiation. As inhibin-A exerts no effect on granulosa cells estrogenic capacity, it seems likely that inhibin-A is either an endocrine modulator of pituitary FSH secretion and/or a paracrine modulator of thecal androgen production, rather than an autocrine modulator of granulosa cells differentiation. Acknowledgements This study was supported by the Jordan University of Science and Technology Grant no. 172/99. References Bartlewski, P.M., Beard, A.P., Cook, S.J., Chandolia, R.K., Honaramooz, A., Rawlings, N.C., 1999. Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oestrous cycle in breeds of sheep differing in proli®cacy. J. Reprod. Fertil. 115, 111±124. Braw-Tal, R., 1994. Expression of mRNA for follistatin and inhibin/activin subunits during follicular growth and atresia. J. Mol. Endocrinol. 13, 253±264. Burger, H.G., Igarashi, M., 1988. Inhibin: de®nition and nomenclature, including related substances. Endocrinology 122, 1701±1702. Campbell, B.K., Gordon, B.M., Tsonis, C.G., Scaramuzzi, R.J., 1995. The effect of acute immuno-neutralisation of inhibin in ewes during the early luteal phase of the oestrous cycle on ovarian hormone secretion and follicular development. J. Endocrinol. 145, 479±490.
F. Shidaifat / Small Ruminant Research 42 (2001) 179±183 Campbell, B.K., Engelhardt, H., McNeilly, A.S., Harkness, L.M., Fukuoka, M., Baird, D.T., 1999. Direct effects of ovine follicular ¯uid on ovarian steroid secretion and expression of markers of cellular differentiation in sheep. J. Reprod. Fertil. 117, 259±269. Carroll, R.S., Kowash, P.M., Lofgren, J.A., Schwall, R.H., Chin, W.W., 1991. In vivo regulation of FSH synthesis by inhibin and activin. Endocrinology. 129, 3299±3304. D'Alessandro, A., Martemucci, G., Iaffaldano, N., 1999. Active immunization with a synthetic fragment of pig inhibin asubunit increases ovulation rate and embryo production in superovulated ewes but season affect its ef®ciency. J. Reprod. Fertil. 115, 185±191. Findlay, J.K., 1993. An update on the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biol. Reprod. 48, 15±23. Findlay, J.K., Roberston, D.M., Clarke, I.J., 1987. In¯uence of dose and route of administration of bovine follicular ¯uid and the suppressive effect of puri®ed bovine inhibin (Mr 31,000) on plasma FSH concentration in ovariectomized ewe. J. Reprod. Fertil. 80, 455±461. Ginther, O.J., Kot, K., Wiltbank, M.C., 1995. Association between emergence of follicular waves and ¯uctuations in FSH concentrations during the oestrous cycle in ewes. Theriogenology 43, 689±703. Groome, N.P., Illingworth, P.J., O'Brien, M., Cooke, I., Ganesan, T.S., Baird, D.T., McNeilly, A.S., 1994. Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin. Endocrinol. 40, 717±723. Hillier, S.G., Yong, E.L., Illingworth, P.J., Baird, D.T., Schwall, R.H., Mason, A.J., 1991. Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells. Mol. Cell. Endocrinol. 75, R1±R6. Huet, C., Monget, P., Pisselet, C., Monniaux, D., 1997. Changes in extracellular matrix component and steroidogenic enzymes
183
during growth and atresia of antral ovarian follicles in the sheep. Biol. Reprod. 56, 1025±1034. Knight, P.G., Feist, S.A., Tannetta, D.S., Bleach, E.C.L., Fowler, P.A., O'Brien, M., Groome, N.P., 1998. Measurement of inhibin-A (a bA dimer) during the oestrus cycle, after manipulation of ovarian activity and during pregnancy in ewe. J. Reprod. Fertil. 113, 159±166. Mayo, K.E., Cerelli, G.M., Spiess, J., Rivier, J., Rosenfeld, M.G., Evans, R.M., Vale, W., 1986. Inhibin A subunit cDNAs from porcine ovary and human placenta. Proc. Natl. Acad. Sci. 83, 5849±5853. Rivier, C., Schwall, R., Mason, A., Burton, L., Vaughan, J., Vale, W., 1991. Effect of recombinant inhibin on luteinizing hormone and follicle-stimulating hormone secretion in the rat. Endocrinology 128, 1548±1554. Souza, C.J.H., Campbell, B.K., Webb, R., Baird, D.T., 1997. Secretion of inhibin A and follicular dynamics throughout the estrous cycle in the sheep with and without the booroola gene (FecB). Endocrinology 138, 5333±5340. Souza, C.J., Campbell, B.K., Baird, D.T., 1998. Follicular waves and concentrations of steroids and inhibin A in ovarian venous blood during the luteal phase of the oestrous cycle in ewes with an ovarian autotransplant. J. Endocrinol. 156, 563± 572. Tisdall, D.J.H., Hudson, N., Smith, P., McNatty, K.P., 1994. Localization of ovine follistatin and a and bA inhibin mRNA in the sheep ovary during the oestrous cycle. J. Mol. Endocrinol. 12, 181±193. Tsonis, C.G., Carson, R.S., Findlay, J.K., 1984. Relationships between aromatase activity, follicular ¯uid oestradiol-17 beta and testosterone concentrations, and diameter and atresia of individual ovine follicles. J. Reprod. Fertil. 72, 153±163. Ying, S.-Y., 1988. Inhibins, activins, and follistatin: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev. 9, 267±293.
Inhibin-A and estradiol content of sheep follicles and their association with follicular diameter F. Shidaifat* Department of Basic Veterinary Medical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030 Irbid, Jordan Accepted 27 June 2001
Abstract This study was conducted to investigate dimeric inhibin-A and estradiol content of sheep antral follicles and their association with follicular size. Follicular content of inhibin-A and estradiol in small, medium and large antral follicles were determined by enzyme immunosorbent assay. The results showed that both estradiol and inhibin-A content of follicular ¯uid increased as the follicular size increased from small to medium and large-sized antral follicles. Although estradiol and inhibinA content were signi®cantly higher, in large antral follicles compared to small and medium-sized follicles, no signi®cant increase was observed between medium and small follicles. These results indicated that estrogenic active follicles are capable of producing parallel quantities of inhibin-A. To investigate a possible local role for inhibin-A on estrogenic capacity of sheep granulosa cells, cells obtained from antral follicles were cultured in a chemically de®ned (RPMI-1640) in the presence or absence of 100 ng/ml inhibin for 24, 48 and 72 h. The results showed that granulosa cells acquire an enhanced ability for estradiol production in the absence of inhibin as the time progresses. Inhibin treatment showed no effect on estradiol production of granulosa cells. As inhibin-A is associated with follicular size and their estradiol content, it exerts no effect on estrogenic capacity of granulosa cells. Probably, inhibin-A exerts its effect on sheep follicular development through endocrine and/or paracrine routes rather than an autocrine route. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Inhibin; Estradiol; Granulosa cells; Follicles
1. Introduction Follicular growth occurs as a series of waves throughout the reproductive cycle of sheep. The emergence of these waves have been shown to be associated with a transient increase in serum concentration of follicle stimulating hormone (FSH), suggesting a major role of FSH in the regulation of sheep follicular development (Bartlewski et al.,
* Tel.: 962-2-7095111; fax: 962-2-7095123. E-mail address: [email protected] (F. Shidaifat).
1999; Ginther et al., 1995; Souza et al., 1998). However, the ®ne tunings of FSH secretion by the pituitary gland and the subsequent monitoring of follicular development require a complex interaction with ovarian factors (Ying, 1988; Findlay, 1993). Of those, inhibin, a gonadal-derived glycoprotein is a heterodimer composed of an a-subunit linked by disul®de bonds to either of two bA or bB subunits to form active dimers known as inhibin-A and inhibin-B, respectively (Burger and Igarashi, 1988). Although, mRNA for a, bA and bB inhibin subunits were found to be expressed speci®cally by the granulosa cells of developing healthy follicles in sheep
0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 2 5 8 - 9
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F. Shidaifat / Small Ruminant Research 42 (2001) 179±183
(Braw-Tal, 1994; Tisdall et al., 1994; Campbell et al., 1999), circulating inhibin-A appears to be the predominant circulating form (Knight et al., 1998). The circulating pattern of inhibin-A and its relation to follicular development, has led to the suggestion that the largest dominant follicle is the main source of circulating inhibin-A (Souza et al., 1997, 1998). Although inhibin is characterized primarily by its ability to attenuate FSH secretion by the pituitary gland (Findlay et al., 1987; Rivier et al., 1991; Carroll et al., 1991), a local role in the regulation of ovarian function has been proposed (Campbell et al., 1995). Therefore, this study was conducted to determine the follicular content of inhibin-A and its association with estradiol and follicular size. In addition, a possible local effect of inhibin-A on granulosa cells differentiation was examined in vitro. 2. Materials and methods 2.1. Inhibin-A assay Follicular ¯uid was obtained from Awassi sheep ovaries, which were transported to the laboratory within 2 h post-mortem. The follicular ¯uid was completely aspirated from 24 individual follicles using a Hamilton syringe. The follicles were classi®ed as small (<3 mm), medium (3±6 mm) and largesized antral follicles (>6 mm). The granulosa cells were then removed by centrifugation for 5 min at approximately 200 g. Inhibin-A dimer concentration was determined by ultra sensitive two-site enzyme immunoassay (EIA) kit (SeroTec, Oxford, UK) according to the method of Groome et al. (1994). The follicular ¯uid was diluted in fetal calf serum and then pretreated with 6% sodium dodecyl sulfate and freshly prepared 6% hydrogen peroxide. The samples were then added to a microplate coated with monoclonal antibodies speci®c for bA subunit of inhibin and incubated overnight. At the end of this incubation period, the plate was then washed, and a second monoclonal antibody speci®c for the a subunit of inhibin was added. The secondary antibody is coupled to alkaline phosphatase. Inhibin dimer was detected by addition of alkaline phosphatase substrate, after the removal of the unreacted material by washing.
2.2. Estradiol 17b assay Follicular ¯uid content of estradiol 17b and that secreted by cultured sheep granulosa cells were evaluated by direct estradiol EIA kits (BioSource, Belgium). Absorbance was measured with Dynatech microplate reader at a wavelength of 450 nm. Awassi sheep ovaries, obtained from a local slaughterhouse, were transported to the laboratory within 2 h of slaughter. The ovaries were collected during September from sheep of 7±9 months old. After soaking in 70% ethanol for approximately 1 min, the ovaries were washed ®ve times with distilled water. Since granulosa cells from medium-sized follicles acquired enhanced aromatase activity and estradiol production, granulosa cells harvested from those follicles were used to evaluate the effect of inhibin-A on estradiol production. Granulosa cells were aspirated from antral follicles (3±5 mm in diameter) using a 20-gauge needle ®tted to a 10 ml syringe. The follicular ¯uid containing the granulosa cells was suspended in RPMI-1640 (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and antibiotic±antimycotic mixture (100 IU penicillin, 100 mg/ml streptomycin, and 250 mg/ml amphotericin) (GIBCO BRL, Grand Island, NY). Approximately 2:0 105 live cells in 1 ml RPMI1640, containing 10% FBS and antibiotic±antimycotic mixture, were initially seeded per well in 24-well culture plate (Costar, Corning, NY) and allowed to grow for 48 h in a humidi®ed incubator at 37 8C, 5% CO2 and 95% air. At the end of this culture period, cells reached about 90% con¯uency as assessed by the inverted microscope. The cells were then washed with RPMI-1640. To determine the effect of recombinant inhibin-A (Mayo et al., 1986) (R&D systems, Minneapolis, MN) on granulosa cells estradiol production, cells were cultured for additional 24, 48 and 72 h in the presence or absence of 100 ng/ml inhibinA. At the end of each treatment period, the media were collected for estradiol evaluation by EIA. Cell treatments were carried out in 1 ml of RPMI-1640 supplemented with 1 mg/ml insulin, 0.67 mg/ml transferrin and 0.55 mg/ml selenium (GIBCO BRL, Grand Island, NY). In addition, 10 mg/ml of low density lipoprotein (Sigma, St. Louis, MO) and 1 mM testosterone were used as substrate for steroid synthesis.
F. Shidaifat / Small Ruminant Research 42 (2001) 179±183
181
Least squares procedures were used to analyse inhibin-A and estradiol content using a model that included ®xed effect due to follicle size (small, medium and large). Signi®cant difference among treatments were based on Bonferroni multiple comparison test. 3. Results Sheep ovaries appeared to contain follicles of different sizes with only one large preovulatory follicle. The mean follicular ¯uid content of inhibin-A is shown in Fig. 1. The results showed a progressive increase in the follicular ¯uid content of inhibin-A as the follicle size increased. The mean follicular ¯uid content of inhibin-A was signi®cantly higher in largesized antral follicles compared to medium- and smallsized antral follicles. Although, inhibin content of medium-sized antral follicles was increased two-fold over that of small-sized antral follicles, no signi®cant difference was observed. This increase in the follicular content of inhibin-A of large antral follicles was associated with a progressive increase in estradiol content. The mean follicular ¯uid content of estradiol is shown in Fig. 2. Large size follicles contained higher concentration of estradiol (P < 0:01) compared to medium and small-sized follicles. Although estradiol content increased as the follicular size increased, the increase in estradiol content was signi®cant in large follicles only. The effect of inhibin-A on sheep granulosa cells estradiol production are presented for six replicate
Fig. 1. Pattern of sheep follicular content of inhibin-A in small, medium and large antral follicles. Inhibin-A follicular ¯uid content of eight individual follicles in each group was presented as the mean S:E:M. Bars with different letters are signi®cantly different.
Fig. 2. Pattern of sheep follicular content of estradiol in small, medium and large antral follicles. Estradiol follicular ¯uid content of eight individual follicles in each group was presented as the mean S:E:M. Bars with different letters are signi®cantly different.
Fig. 3. Time-effect of recombinant inhibin-A on basal estradiol production by cultured sheep granulosa cells. Data presented as the mean S:E:M of six replicate culture wells. Bars with different letters are signi®cantly different.
cultures of each treatment (Fig. 3). This experiment was repeated twice using different pools of granulosa cells harvested from medium-sized follicles. The time-effect of inhibin-A on estradiol production by cultured sheep granulosa cells has been presented. Cultured sheep granulosa cells acquired a progressive and signi®cant increase in their estradiol production on the absence of inhibin-A as the time progress. Inhibin-A treatment (100 ng/ml) of cultured sheep granulosa cells showed no effect on their estradiol production at the time interval tested. 4. Discussion The data presented here shed light on the microenvironment of the developing follicles in term of their
182
F. Shidaifat / Small Ruminant Research 42 (2001) 179±183
estradiol and inhibin content. Results of previous investigations of sheep ovarian follicular dynamics and its relation to circulating inhibin-A and estradiol provided indirect evidence that the granulosa cells of the dominant follicle are the main source of circulating inhibin-A and estradiol (Souza et al., 1997, 1998). The results of this study con®rm that proposal by showing a progressive increase in sheep follicular ¯uid content of dimeric inhibin-A and estradiol as the size of the follicles increased with peak values in the largest antral follicles. These results are consistent with the reported expression pattern of inhibin a, bA subunits (Braw-Tal, 1994, Tisdall et al., 1994; Campbell et al., 1999) and aromatase P450 (Huet et al., 1997; Tsonis et al., 1984) by sheep granulosa cells which have been shown to be stage-dependent and speci®c to granulosa cells of healthy antral follicles, but not to atretic follicles. The parallel changes in the pattern of follicular content of inhibin-A and estradiol re¯ected a close temporal relationship between the initiation and subsequent growth of the antral follicles through the various developmental stages, suggesting that the differentiated growing follicles are the main source of inhibin and estradiol. However, the results of this study indicated that medium and small-sized follicles, which present side by side at the same ovary also produced a substantial amount of both factors suggesting a possible contribution of these follicles to circulating inhibin and estradiol. The increased levels of both estradiol and inhibin-A have been shown to be conversely related to the circulating levels of FSH indicating a negative endocrine effect of inhibin-A on FSH secretion by the pituitary gland (Knight et al., 1998; Souza et al., 1997). However, interference with inhibin effect by immunization, which leads to an increase in the ovulation rate, cast doubts on the endocrine role of inhibin on FSH secretion, suggesting a local alternate pathway through which inhibin-A exerts its effect on follicular development (D'Alessandro et al., 1999; Campbell et al., 1995). Therefore, a possible local regulatory role of inhibin-A on follicular development was examined using granulosa cell culture. Granulosa cells of developing follicles acquired enhanced estrogenic activity as during sequential stages of differentiation. The results of this study provided evidence that the sheep granulosa cells of antral follicles are capable of synthesizing substantial quantities of estra-
diol suggesting a spontaneous differentiation of sheep granulosa cells in vitro. However, inhibin-A exerts no effect on the cultured granulosa cell estrogenic capacity. Since inhibin-A produced by differentiated granulosa cells exerts no effect on their differentiation, it appears that increased inhibin-A production is a result of granulosa cells differentiation. Although its autocrine effect on sheep granulosa cells differentiation seems unlikely, inhibin-A appears to have a paracrine effect on thecal androgen production (Hillier et al., 1991). Thecal androgen serves as an important precursor for estradiol production by granulosa cells which is required to support the high granulosa aromatization activities during the phases of follicular growth and differentiation. In short, the data presented here indicated a positive association of inhibin-A and estradiol content of follicular ¯uid with follicular size suggesting that those factors are markers of follicular development and differentiation. As inhibin-A exerts no effect on granulosa cells estrogenic capacity, it seems likely that inhibin-A is either an endocrine modulator of pituitary FSH secretion and/or a paracrine modulator of thecal androgen production, rather than an autocrine modulator of granulosa cells differentiation. Acknowledgements This study was supported by the Jordan University of Science and Technology Grant no. 172/99. References Bartlewski, P.M., Beard, A.P., Cook, S.J., Chandolia, R.K., Honaramooz, A., Rawlings, N.C., 1999. Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oestrous cycle in breeds of sheep differing in proli®cacy. J. Reprod. Fertil. 115, 111±124. Braw-Tal, R., 1994. Expression of mRNA for follistatin and inhibin/activin subunits during follicular growth and atresia. J. Mol. Endocrinol. 13, 253±264. Burger, H.G., Igarashi, M., 1988. Inhibin: de®nition and nomenclature, including related substances. Endocrinology 122, 1701±1702. Campbell, B.K., Gordon, B.M., Tsonis, C.G., Scaramuzzi, R.J., 1995. The effect of acute immuno-neutralisation of inhibin in ewes during the early luteal phase of the oestrous cycle on ovarian hormone secretion and follicular development. J. Endocrinol. 145, 479±490.
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