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Intragonadal regulation of follicular development and ovulation
SCIENCE ELSEVIER
Animal Reproduction Science 42 (19%) 321-331
Intragonadal regulation of follicular development and ovulation J.K. Findlay a**, A.E. Drummond a, R.C. Fry b aPrince Henry’s Institute of Medical Research, P.O. Box 515.2, Clayton 3168, Vie., Australia b Victorian Institute of Animal Science, Werribee 3030, Vie., Australia
Abstract Ovulation rate, the major determinant of prolificacy, is determined by genotype and influenced by environmental factors and is the culmination of folliculogenesis. Control of folliculogenesis lies with the gonadotropins and local regulatory factors in the ovary such as steroids, cytoklnes and
growth factors. This review summarizes a functional model of folliculogenesis, and discusses evidence pertaining to it and to the regulation of ovulation rate, using data in ewes, cows and rats. Particular emphasis is placed on the roles of local regulators in the acquisition and modulation of responsiveness of ovarian cells to gonadotropins, and their roles in proliferation and differentiation. Two mechanisms of determining multiple ovulation are reviewed and applied to known states of proliflcacy in sheep and cattle. Keywords: Folliculogenesis; Ovulation rate; Gonadotrophins; Local regulatory factors; Responsiveness to gonadotrophin; Differentiation; Proliferation; Sheep; Cows; Rats
1. Introduction Ovulation rate is the major determinant of prolificacy in animals. It is determined by genotype and influenced by environmental factors such as nutrition and photoperiod. An ability to manipulate ovulation rate in domestic animals has commercial significance because of its influence on reproductive performance. The control of ovulation rate lies with the interactions between the pituitary gonadotropins, FSH and LH (and perhaps GH and prolactin in some species) and intraovarian factors such as steroids, cytokines and other growth factors. It has been known for many years that folliculogenesis is dependent, at least in its latter stages, on
??
Corresponding
author: Tel.: +61 3 9550 43%; fax: f61
3 9550 6125.
0378-4320/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SO378-4320(96)014881
322
J.K. Findlay et al./Animal Reproduction Science 42 (1996) 321-331
FSH and LH, and that the very early stages of growth can occur without gonadotropins, implying a role for local factors (Findlay and Risbridger, 1987). The most significant recent advances in our understanding of folliculogenesis have been descriptions of the functional processes which occur during growth and differentiation and how these are controlled by local factors and gonadotropins. This has led to follicle classification systems based on functional rather than morphological criteria (Hirshfield, 1991; Scaramuzzi et al., 1993) which provide a better physiological insight into the control of folliculogenesis, and ultimately ovulation rate. This review summarizes the features of a functional model of folliculogenesis, and discusses evidence pertaining to it and to the regulation of ovulation rate, using data in ewes, cows and rats.
2. A model of folliculogenesis A functional model of folliculogenesis in the ewe has been proposed (Scaramuzzi et al., 1993) based on the physiological processes underlying follicle growth and development, and relating these to the morphological landmarks used in earlier models. The major problem with models based only on morphological criteria, particularly size, is the
I
Pool of Primordial Follicles: essentially quiescent; very little alresia
Committed Follicles: no turning back; low rate of alresia
development may continue in the absence of FSH and LH but gonadotrophins can influence process: some atresia __,____-____---_,_ ___ Gonadotrophm-dependent Folkcles: become atretic if [FSH] < 1 .O ng mL_l ; high rate of atresia
cells express LH receptors; e if (FSH] < 1 .O ng mL-1
Occurs in the presence of an LH surge; else atresia after about 72 h
Fig. 1. A model for folliculax growth in the ewe based on the dependence and sensitivity of the follicles to gonadobopins. An ovarian follicle takes about 180 days to develop from the primordial stage to an ovulatory follicle. The different types of follicles are not drawn to scale or in the correct numerical proportions. The FSH concentrations should be regarded as approximate. From Scaramuzzi et al. (1993) with permission.
J.K. Findlay et al./Animal ReproductionScience42 (1996) 321-331
323
grouping of follicles which may be functionally quite distinct. Some of the sequential physiological changes that are critical for growth and development occur within a single morphological stage which masks both the significance of these changes and the uniqueness of each follicle. The functional model consists of five follicular classes based on their dependency and sensitivity to gonadotropins (Fig. 1). Follicles can be quiescent (i.e. primordial), committed to growth (preantral and antral), ovulatory or atretic. Committed follicles become responsive to gonadotropin, but do not have an absolute dependence on gonadotropin for growth until a later stage. At advanced stages of growth, committed follicles may be gonadotropin sensitive, by which an inappropriate stimulation, particularly by LH, can cause atresia. These classes should not be regarded as absolutely discrete because folliculogenesis is considered to be a continuous process with a hierarchy among follicles according to their developmental status. The assumptions of this model also include; the quiescent nature of primordial follicles until they become committed, follicles that are committed to grow either ovulate or become atretic, functional atresia is an irreversible process and can occur at all stages, only FSH and LH are essential for growth and development, and there is a role for autocrine and paracrine factors in folliculogenesis. The validity of these assumptions is the subject of current research some of which is mentioned below. The extent to which this model applies to species other than the ewe remains to be tested. Some analogies are obvious with the functional model for folliculogenesis in the rat (Hirshfield, 1991), and later in this review we will present data on its application to the cow.
3. Commitment
to growth
Follicles are believed to leave the primordial pool in an ordered sequence, but at an unknown rate, and become irreversibly committed to growth (Findlay and Risbridger, 1987; Hirshfield, 1991; Scaramuzzi et al., 1993). The mechanisms responsible for the initiation of growth of primordial follicles remain an enigma. Gonadotropins are not required, indirectly indicating a role for local regulators. It has been suggested that the primordial follicles commence growth in the order in which they were formed and in relation to their association with the rete ovarii.
4. Responsiveness 4.1. Acquiring
to gonadotropin
responsiveness
to gonadotropin
A key event in folliculogenesis is the acquisition of the capacity of follicular cells to respond to gonadotropin. Responsiveness involves expression of the appropriate receptor and maturation of the post-receptor signal transduction systems.
324
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Granulosa cells of early committed (preantral) follicles express FSH receptors. Without responsiveness to FSH, the follicle cannot proceed to the gonadotropin-dependent stage and would become atretic. There are very few factors known to influence the expression of FSH receptors. In rat granulosa cells, they include FSH itself, CAMP analogs, epidermal growth factor (EGF), transforming growth factor (TGF)-p and activin (Findlay, 1994). The ability of activin to upregulate FSH receptors distinguishes it from insulin-like growth factor-l (IGF-1) which does not increase FSH receptor number. The relative importance of activin and TGF-l3 in upregulating FSH receptors has not been resolved. We have postulated a key role for activin in the acquisition of responsiveness to FSH by granulosa cells of committed follicles (Findlay, 1993). We recently observed a differential response of neonatal rat ovarian cells to FSH in terms of inhibin and progesterone production; while the inhibin response to FSH coincided with increased expression of the FSH receptor on day 8, progesterone was not responsive to FSH even by day 12 (Drummond et al., 1995). These results may be explained by either activation of different signal transduction systems subserving stimulation of inhibin and progesterone, or the expression of different forms of FSH receptor responsible for each of the hormones. We have subsequently shown that treatment of day 12 neonatal ovarian cells with activin renders them responsive to FSH in terms of progesterone production (Drummond, Dyson and Findlay, unpublished observations), further supporting a role for activin in the acquisition of responsiveness to FSH. 4.2. Modulating responsiveness to gonadotropin Many growth factors and cytokines are now known to alter the established responsiveness of theta cells to LH and granulosa cells to LH and FSH. Examples include activin, inhibin, IGF-1, EGF, fibroblast growth factor (FGF), TGF-o and -p, tumor necrosis factor OL,interleukin-1, interferon-y and endothelin. These factors can have negative and/or positive actions, and in some cases, the local factor may even subserve the actions of the gonadotropin in an autocrine or paracrine fashion. For example, activin and IGF-1 may subserve the action of FSH on granulosa cells (Findlay, 1993; Findlay, 1995). Many of the growth factors and cytokines share similar effects on ovarian cells. For example, we have shown that IGF-1, TGF-l3 and activin can all increase steroid and inhibin output by rat granulosa cells in the presence of FSH (Findlay, 1995). Does this simply indicate redundancy in the regulatory systems or is there also something unique about the action(s) of these factors that might indicate an obligatory role for each in folliculogenesis? An interesting and perhaps unique action of activin is its changing autocrine effect with respect to the stage of differentiation of the granulosa cells. As the granulosa cells become more differentiated, activin ceases to stimulate progesterone production and becomes inhibitory (Findlay, 1994). The basis of this change is not known but could involve (a> expression of different types of activin receptor, (b) an effect on maturing steroidogenic pathways, or (c) an increasing influence of follistatin, which binds activin but may also have direct effects of its own. Overall, the balance
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between activin and follistatin and between activin and inhibin may be key events in determining the fate of a follicle. We have proposed that activin has a role in the development and maintenance of healthy estrogenic follicles, preventing premature luteinization, whereas follistatin opposes these effects of activin and promotes luteinization or atresia (Findlay, 1993; Findlay, 1994). Others have drawn a similar hypothesis with respect to the balance between IGF-1 and its binding protein(s) on folliculogenesis (Monget and Monniaux, 1995). Do the growth factors interact with each other to promote folliculogenesis? There is a significant interaction between IGF-1 and TGF-l3, even in the absence of FSH, on progesterone production by rat granulosa cells (Nguyen et al., 1994). This implies that in situations where gonadotropin may be limiting, such as FSH in the preovulatory period in ewes and cows and in committed follicles which are not yet responsive to gonadotropin, intrafollicular production of these growth factors may be sufficient to sustain growth and differentiation. While these local actions have been demonstrated in vitro, few have been demonstrated in vivo. The ewe with an ovarian transplant is one model which allows direct testing of local actions by intraarterial infusion of the test substance. Evidence shows that EGF, TGF-a, basic FGF, inhibin and steroid-free bovine follicular fluid inhibit ovarian function, whereas IGF-1 stimulates ovarian hormone secretion (see Campbell et al., 1995). 4.3. Differentiation versus proliferation Growth of the follicle involves both proliferation and differentiation of theta and granulosa cells. The rate at which these cells divide varies with the stage of follicular development. In the ewe, the mitotic index in granulosa cells peaks in committed follicles of about 0.5 mm diameter and declines as they exceed 1.0 mm. At this time, these follicles acquire an increased capacity to produce steroids and they become increasingly responsive to FSH (Scaramuzzi et al., 1993). How are proliferation and differentiation controlled in the same population of cells? Using serum-free culture conditions which do not result in premature luteinization of sheep and cow granulosa cells, Campbell et al. (1995) have demonstrated both proliferative and estrogenic responses to physiological doses of FSH. However, there was a marked difference in the response of cells from small (under 3.5 mm) and large (over 3.5 mm) antral follicles. Cells from small follicles exhibited maximum proliferative and estrogenic responses at FSH doses between 0.5 and 10 ng ml-‘, with no inhibition at 20 ng ml- ’. In contrast, FSH up to 1 ng ml-’ stimulated estrogen production but not proliferation in cells from large follicles, whereas at higher doses of FSH there was proliferation of cells and depression of estrogen. The mechanism by which FSH has these proliferative effects is not known but could be served by expression of an autocrine growth factor. It will be important to investigate how the growth factors and gonadotropins interact to control the cycle of ovarian cells, and the temporal sequence of expression of the growth factors, their binding proteins, their receptors and their metabolism during folliculogenesis, in order to understand the regulation of proliferation and differentiation.
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5. Mechanisms determining ovulation rate The functional model for folliculogenesis describes growth and development of follicles but does not explain the number of ovulatory follicles present at any time. The number of ovulatory follicles is determined by: the number of gonadotropin-responsive follicles immediately available for transformation to gonadotropin-dependent follicles, the viability of gonadotropin-dependent follicles (i.e. their individual requirements for FSH and the influence of local regulatory factors), and the concentrations of gonadotropins determined by the responsiveness of the hypothalamo-pituitary axis to the inhibitory effects of estradiol and inhibin. Campbell et al. (1995) identified 3 levels of control of development and selection of the ovulatory antral follicle; the gonadotropins for follicle development, production of factors by the ovulatory follicle which suppress development of other follicles through gonadotropin-dependent mechanisms, and intrafollicular factors within the ovulatory follicle which enhance or attenuate the actions of gonadotropins. It follows from this discussion that the effect of a given blood concentration of FSH on a follicle would be highly unpredictable, which may explain why, despite the central importance of FSH for folliculogenesis, there is often a poor correlation between blood concentrations of FSH and ovulation rate. 5.1. Determining ovulation rate in the ewe A mechanism for determination of ovulation rate in sheep (Fig. 2) is based around during which gonadotropin-dependent follicles remain viable and may ovulate. In mono-ovulatory animals, the viability of gonadotropin-dependent follicles is severely restricted by their increasing requirement for FSH and by the inhibitory influence of the ovulatory follicle, which restricts the number of ovulatory follicles to one. In multi-ovulatory animals, there are two mechanisms which could apply. The viability of gonadotropin-dependent follicles could be increased by broadening the ‘window’ for FSH and so decreasing atresia (Mechanism A). Multiple ovulation may also be caused by increasing the number of gonadotropin-dependent follicles available for further development, e.g. by increasing the pool size of gonadotropin-dependent follicles available for development (Mechanism B). This may or may not involve (increased) FSH. Multiple ovulation in Booroola ewes carrying the Fee B gene is thought to operate through Mechanism A, as do artificial increases in ovulation rate in ewes caused by exogenous gonadotropins and immunization against inhibin. Increased ovulation rates in the Romanov ewe and the Inverdale ewe carrying the Fee Xl gene are thought to be examples of Mechanism B. ‘windows’
5.2. Determining ovulation rate in the cow Immunization of heifers against a recombinant ovine OLinhibin (AIB) resulted in an increase in ovulation rate, associated with an increase in FSH but not LH (Hillard et al., 1995). Analysis of the data distinguishing between responders and non-responders to immunisation revealed even greater differences in ovulation rates and FSH levels (Fig. 3). Cows have a large number (up to 30) of small antral follicles, a few (one to four)
J.K. Findlay et al./Animal Reproduction Science 42 (1996) 321-331
327
Mono-ovulatory
Mechanism A
Mechanism B
Functional
Functional
2
0
lime
(days)
180
0
Time (days)
180
Fig. 2. A model for the determination of ovulation rate in the ewe. Follicles at different physiological states and developing in a hierarchal sequence are indicated by the vertical arrows. The hatched areas indicate the ‘windows’ during which gonadotropin-dependent follicles remain viable. In Mechanism A, multiple ovulation rate is brought about by a change in ‘window’ during which gonadotropin-dependent follicles are viable. In Mechanism B, multiple ovulation is caused by an increased rate of throughput of follicles without changes in the ‘window’. Mechanism A is probably more common than Mechanism B. From Scaramuzzi et al. (1993) with permission.
medium follicles and generally one large follicle, corresponding approximately to gonadotropin-responsive, gonadotropin-dependent and ovulatory follicles, respectively. In inhibin-immunized parous cows, the increase in ovulation rate (3 vs. 1) was associated with a 169% increase in the number of large (over 8 mm) follicles and a 42% decrease in small follicles (under 5 mm), with no change in the number in the medium group (Fig. 4) (Hillard et al., 1995). These observations show that to achieve an increased ovulation rate in inhibin-immunized cows, plasma FSH must be maintained for an extended period through the late luteal phase and in the following follicular phase, consistent with Mechanism A. We recorded follicle numbers during repeated oocyte recoveries in heifers (Fry et al., 1994a; Fry et al., 1994b) and calves (Fry et al., 199.5). Animals were subjected to ultrasonically-guided transvaginal oocyte retrieval every 3-4 days for a period of 10 weeks, while receiving either eCG (or FSH), AIB or control. Follicles 2 mm or larger were counted and divided into groups of sizes 2-4 mm (small), 5-9 mm (medium) and 10 mm or over (large), corresponding approximately to gonadotropin-responsive, gonadotropin-dependent and ovulatory follicles, respectively. Treatment of 24month-old heifers with either eCG or AIB to increase peripheral concentrations of FSH resulted in a shift in the distribution of follicles from the small to the medium and large groups, without any increase in total number observed at each collection (Fig. 5). Similar effects of FSH and AIB on follicle numbers were seen in 15-month-old heifers. Therefore, FSH
J.K. Findlay et al./Animal Reproduction Science 42 (1996) 321-331
328
PG 3-
‘;-
E P2-
$ s r” 1 -
-3
-2
-1 Days
0
1
2
3
4
relativeto PG injection
Fig. 3. Plasma FSH concentrations (least square means f SEM) in ten control (0 1, 18 inhibin-immune heifers with ovulation rates over 1 (responders, ?? ), and 20 inhibin-immune heifers with single ovulations (non-responders, 0, mean only). Overall FSH concenmtions: responders > non-responders > control (P < 0.001). From Hillard et al. (1995).
does not influence the entry of follicles into groups 2 mm or larger in heifers, but facilitates growth of follicles through the small group leading to a greater proportion of ovulatory follicles, consistent with Mechanism A. The existence in all animals, including controls, of follicles up to 16 mm only 3 days after the previous retrieval suggests that FSH does not change the maximum rate at which follicles are capable of growing (3-4 mm day- ’1, rather it changes the number growing at higher rates. In contrast to FSH, bovine somatotropin (bST) treatment of heifers increased the number of small follicles and consequently increased the total number of size 2 mm or larger (Gong and Webb, 1993), consistent with Mechanism B. The increased number of follicles in the pool was not reflected in an increase in ovulation rate unless eCG was combined with bST treatment. Treatment of 5-month-old calves with either FSH or AIB shifted more follicles into the larger size group, similar to that seen in heifers (Fig, 6). When the data were examined in terms of time after starting FSH treatment (Fig. 71, it was observed that total follicle numbers also increased but only at the early collections. Thereafter, the total follicle numbers returned to pre-FSH levels but the distributions favored large follicles. This would be consistent with Mechanisms B operating early in the experiment and Mechanism A operating throughout. Increased follicle numbers were not observed in early collections in the AIB group making it consistent with Mechanism A only. Maximum growth rates of follicles (up to 4 mm day-’ ) were similar in calves and
J.K. Findiay et al./Animal
Reproduction
Science 42 (1996) 321-331
329
*
T
T
T *
Ovulation rate
Large follicles
s
Medium follicles
fo
~ Ill
les
Fig. 4. Mean f SEM ovulation rates and numbers of large (over 8 mm), medium (S-8 mm) and small (under 5 mm) diameter follicles in the 11 days before ovulation until the day after ovulation in eight control ( 0 ) and 15 inhibin-immune cows ( W). Asterisks indicate significant difference compared with controls (P < 0.05). From Hillard et al. ( 1995).
25
24monthheifm 1
20
0
Control
•j
PMSG
??AIB
I 3 15 = e % . 10 z" 5 0 2-4 mm
5+ mm
Total number
Fig. 5. Average numbers of small (2-4 mm), medium plus large (5 + mm) and total follicles in control, eCG (PMSG)-treated and inhibin-immune (AIB) 24month-old heifers over a IO-week period.
J.K. Findlay et al./ Animal Reproduction Science 42 (1996) 321-331
330
351
5 month calves 0
Control
? ?FSH ??AIB
2-4 mm
5+ mm
Total number
Fig. 6. Average numbers of small (2-4 mm), medium plus large (5 + mm) and total follicles in control, FSH-treated and inhibin-immune (AIB) 5-month-old calves over a 3-week period.
heifers, but treatment with bST had no effect on follicle number or distribution in the calves, unlike the heifers (Gong and Webb, 1993). It is concluded that although FSH influences follicle growth in the calf and heifer, there are factors influencing folliculogenesis in the calf which are different from the heifer. In particular, the relatively senescent calf ovary appears to be initially more responsive to gonadotropin stimulation than the heifer ovary, an effect which disappears with the emergence of many large follicles after stimulation. This would be consistent with local factors produced by large follicles influencing the growth of smaller follicles (Findlay and Risbridger, 1987). The experiments also showed that it is possible to harvest around ten oocytes every 3-4 days from both calves and heifers for use in IVF/IVM protocols.
60
5monlhcahfestreaMwithFSH
? ?Pre FSH ? ?Post FSH 1 ??Post FSH6
50 g 40 .z h 30 b p 20 IO 0 2-4 mm
5+ mm
Total number
Fig. 7. Average numbers of small (2-4 mm), medium plus large (5 + mm) and total follicles in control and FSH-treated 5-month-old calves before the fmst FSH treatment (Pre FSH), and 2 days (Post FSH 1) and 19 days (Post FSH 6) later with continuing FSH treatment.
J.K. Findlay et al. /Animal Reproduction Science 42 (1996) 321-331
331
Acknowledgements The financial support of the NH and MRC of Australia, the Dairy Research Corporation and Biotech Australia Pty Ltd is gratefully acknowledged. Our thanks to Faye Coates and Sue Pan&ridge for help with the manuscript.
References Campbell, B.K., Scaramuzzi, R.J. and Webb, R., 1995. Control of antral follicle development and selection in sheep and cattle. J. Reprod. Fertil. Suppl., 49: 335-350. Drummond, A.E., Dyson, M. and Findlay, J.K., 1995. Differential response of dispersed neonatal rat ovaries to FSH stimulation. Proc. Aust. Endocrinol. Sot., 38: 56. 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., 1994. Peripheral and local regulators of folliculogenesis. Reprod. Fertil. Dev., 6: 127- 139. Findlay, J.K., 1995. Is the intraovarian IGF system a mediator of growth hormone action? In: E.Y. Adashi and M.O. Thomer (Editors), The Somatotrophic Axis and the Reproductive Process in Health and Disease. Serono Symposium, Springer, New York, pp. 202-211. Fmdlay, J.K. and Risbridger, G.P., 1987. Intragonadal control mechanisms. In: H.G. Burger (Editor), Bail&es Clinical Endocrinology and Metabolism. W.B. Saunders, London, pp. 223-243. Fry, R.C., Simpson, T.L., Squires, T.J., Parr, R.A. and Damanik, R.M., 1994a. Factors affecting transvaginal oocyte pick up in heifers. Theriogenology, 41: 197. Fry, R.C., Simpson, T.L., Squires, T.J. and Findlay, J.K., 1994b. Follicular growth and oocyte collection in heifers immunised against inhibin. Proc. Aust. Sot. Reprod. Biol., 26: 24. Fry, R.C., Simpson, T.L., Squires, T.J., Harris, S.G. and Findlay, J.K., 1995. The effect of FSH, AIB and bST on follicular growth and oocyte recovery in young calves. Proc. Aust. Sot. Reprod. Biol., 27: 76. Gong, J.G. and Webb, R., 1993. The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers. J. Reprod. Fertil., 97: 247-254. Hillard, M.A., Wilkins, J.F., Cummins, L.J., Bindon, B.M., Tsonis, C.G., Fmdlay, J.K. and O’Shea, T., 1995. Immunological manipulation of ovulation rate for twinning in cattle. J. Reprod. Fertil. Suppl., 49: 35 l-364. Hirshfield, A.N., 1991. Development of follicles in the mammalian ovary. Int. Rev. Cytol., 124: 43- 101. Monget, P. and Monniaux, D., 1995. Growth factors and control of folliculogenesis. J. Reprod. Fertil. Suppl., 49: 321-333. Nguyen, T., Drummond, A.E., Dyson, M. and Findlay, J.K., 1994. Synergistic effects of transforming growth factor-6 (TGF-B) and insulin-like growth factor-l (IGF-1) on granulosa cell function. hoc. Aust. SOC. Med. Res., 23: 4-6. Scaramuzzi, R.J., Adams, N.R., Baird, D.T., Campbell, B.K., Downing, J.A., Findlay, J.K., Henderson, K.M., Martin, G.B.. McNatty, K.P., McNeilly, A.S. and Tsonis, C.G.. 1993. A model for follicle selection and the determination of ovulation rate in the ewe. Reprod. Fertil. Dev., 5: 459-478.
Animal Reproduction Science 42 (19%) 321-331
Intragonadal regulation of follicular development and ovulation J.K. Findlay a**, A.E. Drummond a, R.C. Fry b aPrince Henry’s Institute of Medical Research, P.O. Box 515.2, Clayton 3168, Vie., Australia b Victorian Institute of Animal Science, Werribee 3030, Vie., Australia
Abstract Ovulation rate, the major determinant of prolificacy, is determined by genotype and influenced by environmental factors and is the culmination of folliculogenesis. Control of folliculogenesis lies with the gonadotropins and local regulatory factors in the ovary such as steroids, cytoklnes and
growth factors. This review summarizes a functional model of folliculogenesis, and discusses evidence pertaining to it and to the regulation of ovulation rate, using data in ewes, cows and rats. Particular emphasis is placed on the roles of local regulators in the acquisition and modulation of responsiveness of ovarian cells to gonadotropins, and their roles in proliferation and differentiation. Two mechanisms of determining multiple ovulation are reviewed and applied to known states of proliflcacy in sheep and cattle. Keywords: Folliculogenesis; Ovulation rate; Gonadotrophins; Local regulatory factors; Responsiveness to gonadotrophin; Differentiation; Proliferation; Sheep; Cows; Rats
1. Introduction Ovulation rate is the major determinant of prolificacy in animals. It is determined by genotype and influenced by environmental factors such as nutrition and photoperiod. An ability to manipulate ovulation rate in domestic animals has commercial significance because of its influence on reproductive performance. The control of ovulation rate lies with the interactions between the pituitary gonadotropins, FSH and LH (and perhaps GH and prolactin in some species) and intraovarian factors such as steroids, cytokines and other growth factors. It has been known for many years that folliculogenesis is dependent, at least in its latter stages, on
??
Corresponding
author: Tel.: +61 3 9550 43%; fax: f61
3 9550 6125.
0378-4320/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SO378-4320(96)014881
322
J.K. Findlay et al./Animal Reproduction Science 42 (1996) 321-331
FSH and LH, and that the very early stages of growth can occur without gonadotropins, implying a role for local factors (Findlay and Risbridger, 1987). The most significant recent advances in our understanding of folliculogenesis have been descriptions of the functional processes which occur during growth and differentiation and how these are controlled by local factors and gonadotropins. This has led to follicle classification systems based on functional rather than morphological criteria (Hirshfield, 1991; Scaramuzzi et al., 1993) which provide a better physiological insight into the control of folliculogenesis, and ultimately ovulation rate. This review summarizes the features of a functional model of folliculogenesis, and discusses evidence pertaining to it and to the regulation of ovulation rate, using data in ewes, cows and rats.
2. A model of folliculogenesis A functional model of folliculogenesis in the ewe has been proposed (Scaramuzzi et al., 1993) based on the physiological processes underlying follicle growth and development, and relating these to the morphological landmarks used in earlier models. The major problem with models based only on morphological criteria, particularly size, is the
I
Pool of Primordial Follicles: essentially quiescent; very little alresia
Committed Follicles: no turning back; low rate of alresia
development may continue in the absence of FSH and LH but gonadotrophins can influence process: some atresia __,____-____---_,_ ___ Gonadotrophm-dependent Folkcles: become atretic if [FSH] < 1 .O ng mL_l ; high rate of atresia
cells express LH receptors; e if (FSH] < 1 .O ng mL-1
Occurs in the presence of an LH surge; else atresia after about 72 h
Fig. 1. A model for folliculax growth in the ewe based on the dependence and sensitivity of the follicles to gonadobopins. An ovarian follicle takes about 180 days to develop from the primordial stage to an ovulatory follicle. The different types of follicles are not drawn to scale or in the correct numerical proportions. The FSH concentrations should be regarded as approximate. From Scaramuzzi et al. (1993) with permission.
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323
grouping of follicles which may be functionally quite distinct. Some of the sequential physiological changes that are critical for growth and development occur within a single morphological stage which masks both the significance of these changes and the uniqueness of each follicle. The functional model consists of five follicular classes based on their dependency and sensitivity to gonadotropins (Fig. 1). Follicles can be quiescent (i.e. primordial), committed to growth (preantral and antral), ovulatory or atretic. Committed follicles become responsive to gonadotropin, but do not have an absolute dependence on gonadotropin for growth until a later stage. At advanced stages of growth, committed follicles may be gonadotropin sensitive, by which an inappropriate stimulation, particularly by LH, can cause atresia. These classes should not be regarded as absolutely discrete because folliculogenesis is considered to be a continuous process with a hierarchy among follicles according to their developmental status. The assumptions of this model also include; the quiescent nature of primordial follicles until they become committed, follicles that are committed to grow either ovulate or become atretic, functional atresia is an irreversible process and can occur at all stages, only FSH and LH are essential for growth and development, and there is a role for autocrine and paracrine factors in folliculogenesis. The validity of these assumptions is the subject of current research some of which is mentioned below. The extent to which this model applies to species other than the ewe remains to be tested. Some analogies are obvious with the functional model for folliculogenesis in the rat (Hirshfield, 1991), and later in this review we will present data on its application to the cow.
3. Commitment
to growth
Follicles are believed to leave the primordial pool in an ordered sequence, but at an unknown rate, and become irreversibly committed to growth (Findlay and Risbridger, 1987; Hirshfield, 1991; Scaramuzzi et al., 1993). The mechanisms responsible for the initiation of growth of primordial follicles remain an enigma. Gonadotropins are not required, indirectly indicating a role for local regulators. It has been suggested that the primordial follicles commence growth in the order in which they were formed and in relation to their association with the rete ovarii.
4. Responsiveness 4.1. Acquiring
to gonadotropin
responsiveness
to gonadotropin
A key event in folliculogenesis is the acquisition of the capacity of follicular cells to respond to gonadotropin. Responsiveness involves expression of the appropriate receptor and maturation of the post-receptor signal transduction systems.
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Granulosa cells of early committed (preantral) follicles express FSH receptors. Without responsiveness to FSH, the follicle cannot proceed to the gonadotropin-dependent stage and would become atretic. There are very few factors known to influence the expression of FSH receptors. In rat granulosa cells, they include FSH itself, CAMP analogs, epidermal growth factor (EGF), transforming growth factor (TGF)-p and activin (Findlay, 1994). The ability of activin to upregulate FSH receptors distinguishes it from insulin-like growth factor-l (IGF-1) which does not increase FSH receptor number. The relative importance of activin and TGF-l3 in upregulating FSH receptors has not been resolved. We have postulated a key role for activin in the acquisition of responsiveness to FSH by granulosa cells of committed follicles (Findlay, 1993). We recently observed a differential response of neonatal rat ovarian cells to FSH in terms of inhibin and progesterone production; while the inhibin response to FSH coincided with increased expression of the FSH receptor on day 8, progesterone was not responsive to FSH even by day 12 (Drummond et al., 1995). These results may be explained by either activation of different signal transduction systems subserving stimulation of inhibin and progesterone, or the expression of different forms of FSH receptor responsible for each of the hormones. We have subsequently shown that treatment of day 12 neonatal ovarian cells with activin renders them responsive to FSH in terms of progesterone production (Drummond, Dyson and Findlay, unpublished observations), further supporting a role for activin in the acquisition of responsiveness to FSH. 4.2. Modulating responsiveness to gonadotropin Many growth factors and cytokines are now known to alter the established responsiveness of theta cells to LH and granulosa cells to LH and FSH. Examples include activin, inhibin, IGF-1, EGF, fibroblast growth factor (FGF), TGF-o and -p, tumor necrosis factor OL,interleukin-1, interferon-y and endothelin. These factors can have negative and/or positive actions, and in some cases, the local factor may even subserve the actions of the gonadotropin in an autocrine or paracrine fashion. For example, activin and IGF-1 may subserve the action of FSH on granulosa cells (Findlay, 1993; Findlay, 1995). Many of the growth factors and cytokines share similar effects on ovarian cells. For example, we have shown that IGF-1, TGF-l3 and activin can all increase steroid and inhibin output by rat granulosa cells in the presence of FSH (Findlay, 1995). Does this simply indicate redundancy in the regulatory systems or is there also something unique about the action(s) of these factors that might indicate an obligatory role for each in folliculogenesis? An interesting and perhaps unique action of activin is its changing autocrine effect with respect to the stage of differentiation of the granulosa cells. As the granulosa cells become more differentiated, activin ceases to stimulate progesterone production and becomes inhibitory (Findlay, 1994). The basis of this change is not known but could involve (a> expression of different types of activin receptor, (b) an effect on maturing steroidogenic pathways, or (c) an increasing influence of follistatin, which binds activin but may also have direct effects of its own. Overall, the balance
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between activin and follistatin and between activin and inhibin may be key events in determining the fate of a follicle. We have proposed that activin has a role in the development and maintenance of healthy estrogenic follicles, preventing premature luteinization, whereas follistatin opposes these effects of activin and promotes luteinization or atresia (Findlay, 1993; Findlay, 1994). Others have drawn a similar hypothesis with respect to the balance between IGF-1 and its binding protein(s) on folliculogenesis (Monget and Monniaux, 1995). Do the growth factors interact with each other to promote folliculogenesis? There is a significant interaction between IGF-1 and TGF-l3, even in the absence of FSH, on progesterone production by rat granulosa cells (Nguyen et al., 1994). This implies that in situations where gonadotropin may be limiting, such as FSH in the preovulatory period in ewes and cows and in committed follicles which are not yet responsive to gonadotropin, intrafollicular production of these growth factors may be sufficient to sustain growth and differentiation. While these local actions have been demonstrated in vitro, few have been demonstrated in vivo. The ewe with an ovarian transplant is one model which allows direct testing of local actions by intraarterial infusion of the test substance. Evidence shows that EGF, TGF-a, basic FGF, inhibin and steroid-free bovine follicular fluid inhibit ovarian function, whereas IGF-1 stimulates ovarian hormone secretion (see Campbell et al., 1995). 4.3. Differentiation versus proliferation Growth of the follicle involves both proliferation and differentiation of theta and granulosa cells. The rate at which these cells divide varies with the stage of follicular development. In the ewe, the mitotic index in granulosa cells peaks in committed follicles of about 0.5 mm diameter and declines as they exceed 1.0 mm. At this time, these follicles acquire an increased capacity to produce steroids and they become increasingly responsive to FSH (Scaramuzzi et al., 1993). How are proliferation and differentiation controlled in the same population of cells? Using serum-free culture conditions which do not result in premature luteinization of sheep and cow granulosa cells, Campbell et al. (1995) have demonstrated both proliferative and estrogenic responses to physiological doses of FSH. However, there was a marked difference in the response of cells from small (under 3.5 mm) and large (over 3.5 mm) antral follicles. Cells from small follicles exhibited maximum proliferative and estrogenic responses at FSH doses between 0.5 and 10 ng ml-‘, with no inhibition at 20 ng ml- ’. In contrast, FSH up to 1 ng ml-’ stimulated estrogen production but not proliferation in cells from large follicles, whereas at higher doses of FSH there was proliferation of cells and depression of estrogen. The mechanism by which FSH has these proliferative effects is not known but could be served by expression of an autocrine growth factor. It will be important to investigate how the growth factors and gonadotropins interact to control the cycle of ovarian cells, and the temporal sequence of expression of the growth factors, their binding proteins, their receptors and their metabolism during folliculogenesis, in order to understand the regulation of proliferation and differentiation.
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5. Mechanisms determining ovulation rate The functional model for folliculogenesis describes growth and development of follicles but does not explain the number of ovulatory follicles present at any time. The number of ovulatory follicles is determined by: the number of gonadotropin-responsive follicles immediately available for transformation to gonadotropin-dependent follicles, the viability of gonadotropin-dependent follicles (i.e. their individual requirements for FSH and the influence of local regulatory factors), and the concentrations of gonadotropins determined by the responsiveness of the hypothalamo-pituitary axis to the inhibitory effects of estradiol and inhibin. Campbell et al. (1995) identified 3 levels of control of development and selection of the ovulatory antral follicle; the gonadotropins for follicle development, production of factors by the ovulatory follicle which suppress development of other follicles through gonadotropin-dependent mechanisms, and intrafollicular factors within the ovulatory follicle which enhance or attenuate the actions of gonadotropins. It follows from this discussion that the effect of a given blood concentration of FSH on a follicle would be highly unpredictable, which may explain why, despite the central importance of FSH for folliculogenesis, there is often a poor correlation between blood concentrations of FSH and ovulation rate. 5.1. Determining ovulation rate in the ewe A mechanism for determination of ovulation rate in sheep (Fig. 2) is based around during which gonadotropin-dependent follicles remain viable and may ovulate. In mono-ovulatory animals, the viability of gonadotropin-dependent follicles is severely restricted by their increasing requirement for FSH and by the inhibitory influence of the ovulatory follicle, which restricts the number of ovulatory follicles to one. In multi-ovulatory animals, there are two mechanisms which could apply. The viability of gonadotropin-dependent follicles could be increased by broadening the ‘window’ for FSH and so decreasing atresia (Mechanism A). Multiple ovulation may also be caused by increasing the number of gonadotropin-dependent follicles available for further development, e.g. by increasing the pool size of gonadotropin-dependent follicles available for development (Mechanism B). This may or may not involve (increased) FSH. Multiple ovulation in Booroola ewes carrying the Fee B gene is thought to operate through Mechanism A, as do artificial increases in ovulation rate in ewes caused by exogenous gonadotropins and immunization against inhibin. Increased ovulation rates in the Romanov ewe and the Inverdale ewe carrying the Fee Xl gene are thought to be examples of Mechanism B. ‘windows’
5.2. Determining ovulation rate in the cow Immunization of heifers against a recombinant ovine OLinhibin (AIB) resulted in an increase in ovulation rate, associated with an increase in FSH but not LH (Hillard et al., 1995). Analysis of the data distinguishing between responders and non-responders to immunisation revealed even greater differences in ovulation rates and FSH levels (Fig. 3). Cows have a large number (up to 30) of small antral follicles, a few (one to four)
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Mono-ovulatory
Mechanism A
Mechanism B
Functional
Functional
2
0
lime
(days)
180
0
Time (days)
180
Fig. 2. A model for the determination of ovulation rate in the ewe. Follicles at different physiological states and developing in a hierarchal sequence are indicated by the vertical arrows. The hatched areas indicate the ‘windows’ during which gonadotropin-dependent follicles remain viable. In Mechanism A, multiple ovulation rate is brought about by a change in ‘window’ during which gonadotropin-dependent follicles are viable. In Mechanism B, multiple ovulation is caused by an increased rate of throughput of follicles without changes in the ‘window’. Mechanism A is probably more common than Mechanism B. From Scaramuzzi et al. (1993) with permission.
medium follicles and generally one large follicle, corresponding approximately to gonadotropin-responsive, gonadotropin-dependent and ovulatory follicles, respectively. In inhibin-immunized parous cows, the increase in ovulation rate (3 vs. 1) was associated with a 169% increase in the number of large (over 8 mm) follicles and a 42% decrease in small follicles (under 5 mm), with no change in the number in the medium group (Fig. 4) (Hillard et al., 1995). These observations show that to achieve an increased ovulation rate in inhibin-immunized cows, plasma FSH must be maintained for an extended period through the late luteal phase and in the following follicular phase, consistent with Mechanism A. We recorded follicle numbers during repeated oocyte recoveries in heifers (Fry et al., 1994a; Fry et al., 1994b) and calves (Fry et al., 199.5). Animals were subjected to ultrasonically-guided transvaginal oocyte retrieval every 3-4 days for a period of 10 weeks, while receiving either eCG (or FSH), AIB or control. Follicles 2 mm or larger were counted and divided into groups of sizes 2-4 mm (small), 5-9 mm (medium) and 10 mm or over (large), corresponding approximately to gonadotropin-responsive, gonadotropin-dependent and ovulatory follicles, respectively. Treatment of 24month-old heifers with either eCG or AIB to increase peripheral concentrations of FSH resulted in a shift in the distribution of follicles from the small to the medium and large groups, without any increase in total number observed at each collection (Fig. 5). Similar effects of FSH and AIB on follicle numbers were seen in 15-month-old heifers. Therefore, FSH
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PG 3-
‘;-
E P2-
$ s r” 1 -
-3
-2
-1 Days
0
1
2
3
4
relativeto PG injection
Fig. 3. Plasma FSH concentrations (least square means f SEM) in ten control (0 1, 18 inhibin-immune heifers with ovulation rates over 1 (responders, ?? ), and 20 inhibin-immune heifers with single ovulations (non-responders, 0, mean only). Overall FSH concenmtions: responders > non-responders > control (P < 0.001). From Hillard et al. (1995).
does not influence the entry of follicles into groups 2 mm or larger in heifers, but facilitates growth of follicles through the small group leading to a greater proportion of ovulatory follicles, consistent with Mechanism A. The existence in all animals, including controls, of follicles up to 16 mm only 3 days after the previous retrieval suggests that FSH does not change the maximum rate at which follicles are capable of growing (3-4 mm day- ’1, rather it changes the number growing at higher rates. In contrast to FSH, bovine somatotropin (bST) treatment of heifers increased the number of small follicles and consequently increased the total number of size 2 mm or larger (Gong and Webb, 1993), consistent with Mechanism B. The increased number of follicles in the pool was not reflected in an increase in ovulation rate unless eCG was combined with bST treatment. Treatment of 5-month-old calves with either FSH or AIB shifted more follicles into the larger size group, similar to that seen in heifers (Fig, 6). When the data were examined in terms of time after starting FSH treatment (Fig. 71, it was observed that total follicle numbers also increased but only at the early collections. Thereafter, the total follicle numbers returned to pre-FSH levels but the distributions favored large follicles. This would be consistent with Mechanisms B operating early in the experiment and Mechanism A operating throughout. Increased follicle numbers were not observed in early collections in the AIB group making it consistent with Mechanism A only. Maximum growth rates of follicles (up to 4 mm day-’ ) were similar in calves and
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*
T
T
T *
Ovulation rate
Large follicles
s
Medium follicles
fo
~ Ill
les
Fig. 4. Mean f SEM ovulation rates and numbers of large (over 8 mm), medium (S-8 mm) and small (under 5 mm) diameter follicles in the 11 days before ovulation until the day after ovulation in eight control ( 0 ) and 15 inhibin-immune cows ( W). Asterisks indicate significant difference compared with controls (P < 0.05). From Hillard et al. ( 1995).
25
24monthheifm 1
20
0
Control
•j
PMSG
??AIB
I 3 15 = e % . 10 z" 5 0 2-4 mm
5+ mm
Total number
Fig. 5. Average numbers of small (2-4 mm), medium plus large (5 + mm) and total follicles in control, eCG (PMSG)-treated and inhibin-immune (AIB) 24month-old heifers over a IO-week period.
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351
5 month calves 0
Control
? ?FSH ??AIB
2-4 mm
5+ mm
Total number
Fig. 6. Average numbers of small (2-4 mm), medium plus large (5 + mm) and total follicles in control, FSH-treated and inhibin-immune (AIB) 5-month-old calves over a 3-week period.
heifers, but treatment with bST had no effect on follicle number or distribution in the calves, unlike the heifers (Gong and Webb, 1993). It is concluded that although FSH influences follicle growth in the calf and heifer, there are factors influencing folliculogenesis in the calf which are different from the heifer. In particular, the relatively senescent calf ovary appears to be initially more responsive to gonadotropin stimulation than the heifer ovary, an effect which disappears with the emergence of many large follicles after stimulation. This would be consistent with local factors produced by large follicles influencing the growth of smaller follicles (Findlay and Risbridger, 1987). The experiments also showed that it is possible to harvest around ten oocytes every 3-4 days from both calves and heifers for use in IVF/IVM protocols.
60
5monlhcahfestreaMwithFSH
? ?Pre FSH ? ?Post FSH 1 ??Post FSH6
50 g 40 .z h 30 b p 20 IO 0 2-4 mm
5+ mm
Total number
Fig. 7. Average numbers of small (2-4 mm), medium plus large (5 + mm) and total follicles in control and FSH-treated 5-month-old calves before the fmst FSH treatment (Pre FSH), and 2 days (Post FSH 1) and 19 days (Post FSH 6) later with continuing FSH treatment.
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Acknowledgements The financial support of the NH and MRC of Australia, the Dairy Research Corporation and Biotech Australia Pty Ltd is gratefully acknowledged. Our thanks to Faye Coates and Sue Pan&ridge for help with the manuscript.
References Campbell, B.K., Scaramuzzi, R.J. and Webb, R., 1995. Control of antral follicle development and selection in sheep and cattle. J. Reprod. Fertil. Suppl., 49: 335-350. Drummond, A.E., Dyson, M. and Findlay, J.K., 1995. Differential response of dispersed neonatal rat ovaries to FSH stimulation. Proc. Aust. Endocrinol. Sot., 38: 56. 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., 1994. Peripheral and local regulators of folliculogenesis. Reprod. Fertil. Dev., 6: 127- 139. Findlay, J.K., 1995. Is the intraovarian IGF system a mediator of growth hormone action? In: E.Y. Adashi and M.O. Thomer (Editors), The Somatotrophic Axis and the Reproductive Process in Health and Disease. Serono Symposium, Springer, New York, pp. 202-211. Fmdlay, J.K. and Risbridger, G.P., 1987. Intragonadal control mechanisms. In: H.G. Burger (Editor), Bail&es Clinical Endocrinology and Metabolism. W.B. Saunders, London, pp. 223-243. Fry, R.C., Simpson, T.L., Squires, T.J., Parr, R.A. and Damanik, R.M., 1994a. Factors affecting transvaginal oocyte pick up in heifers. Theriogenology, 41: 197. Fry, R.C., Simpson, T.L., Squires, T.J. and Findlay, J.K., 1994b. Follicular growth and oocyte collection in heifers immunised against inhibin. Proc. Aust. Sot. Reprod. Biol., 26: 24. Fry, R.C., Simpson, T.L., Squires, T.J., Harris, S.G. and Findlay, J.K., 1995. The effect of FSH, AIB and bST on follicular growth and oocyte recovery in young calves. Proc. Aust. Sot. Reprod. Biol., 27: 76. Gong, J.G. and Webb, R., 1993. The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers. J. Reprod. Fertil., 97: 247-254. Hillard, M.A., Wilkins, J.F., Cummins, L.J., Bindon, B.M., Tsonis, C.G., Fmdlay, J.K. and O’Shea, T., 1995. Immunological manipulation of ovulation rate for twinning in cattle. J. Reprod. Fertil. Suppl., 49: 35 l-364. Hirshfield, A.N., 1991. Development of follicles in the mammalian ovary. Int. Rev. Cytol., 124: 43- 101. Monget, P. and Monniaux, D., 1995. Growth factors and control of folliculogenesis. J. Reprod. Fertil. Suppl., 49: 321-333. Nguyen, T., Drummond, A.E., Dyson, M. and Findlay, J.K., 1994. Synergistic effects of transforming growth factor-6 (TGF-B) and insulin-like growth factor-l (IGF-1) on granulosa cell function. hoc. Aust. SOC. Med. Res., 23: 4-6. Scaramuzzi, R.J., Adams, N.R., Baird, D.T., Campbell, B.K., Downing, J.A., Findlay, J.K., Henderson, K.M., Martin, G.B.. McNatty, K.P., McNeilly, A.S. and Tsonis, C.G.. 1993. A model for follicle selection and the determination of ovulation rate in the ewe. Reprod. Fertil. Dev., 5: 459-478.