The N-terminal peptide of the inhibin α subunit

not known if posttranslational cleavage of the a precursor at the Arg-Arg sites also occurs intracellularly. Expression of the inhibin-a

The N-terminal Peptide of the Inhibin CYSubunit What Are Its Endocrine and Paracrine Roles? Darryl L. Russell and Jock K. Findlay CLNinhibin (molecular mass 23/24 kD) is present in the pro-cxN-olC subunit of inhibin and can be released by cleavage at the flanking arginine residues during posttranslational processing. Although the aN protein isolated from bovine follicular fluid has no inhibinlike (FSH suppressing) activity, cxN is present in high molecular weight forms of biologically active inhibin found in follicular fluid and plasma. cxN may modify the biological activity of inhibin by influencing its half-life or access to its receptor. olN may also play a role in regulating fertility through a local action on ovulation by the ovary that is independent of the actions of inhibin. The evidence suggests a unique physiological significance for the precursor peptides of the inhibin-cw subunit in both the endocrine and paracrine control of fertility. (Trends Endocrinol Metab

??

1995;6:305-3

11).

Expression and Forms of aN

give 31/32-kD inhibin,

Inhibin is a glycoprotein hormone consisting of two dissimilar, disulfide-linked subunits, termed cxand p, which inhibits the synthesis and/or secretion of FSH (Burger 1988). The a subunit of inhibin is encoded by a single gene, whereas two genes encode the different, but highly homologous, PA and PB subunits (Mason et al. 1985, Forage et al. 1986). The prepro-a subunit is thought to undergo posttranslational processing by proteolytic cleavage, giving rise to the following peptides; a 20-kD carboxy terminal aC, a 23/24-kD aN, a 43-kD aN-aC, and/or a pro-& ( 6+20 kD), none of which have inhibinlike FSH suppressing activity [see Findlay et al. (1991)] (Figure 1). Disulfide linkage of the 15kD p (A or B) subunit with either 43-kD aNaC, to give 58-kD inhibin, or 20-kDaC, to

active molecules activity.

residues and a potential N-linked glycosylation site. The aN portion of the inhibin-a gene is split between the two exons of the cx gene, suggesting

TEM Vol.6,No.9110, 1995

01995,

that in-

dependent expression of the aN moiety is not possible, but it is likely to be exas part of the a precursor

and

subsequently released by proteolytic cleavage. So, for every inhibin molecule formed, there will be one aN molecule either retained with the inhibin or released into follicular fluid or plasma, Although aN has been isolated from follicular fluid after acidification

Darryl L. Russell and Jock K. Findlay are at Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia; Darryl L. Russell is also currently at the Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.

superovulated compared to normal women (Lambert-Masserlian et al. 1994) and the inhibin-a subunit is differentially processed during development of the hamster testis (Touhimaa et al. 1993), giving substance to the proposition that the inhibin-a subunit or its components may have activities quite distinct from the systemic endocrine role of inhibin. For example, it has been shown that the pro-regions of activin, a dimer of the inhibin /3 subunits, and transforming growth factor (TGF)-PI, aid in the folding, disulfide bond formation and export of their respective homodimers (Gray and Mason 1990). Similar studies to demonstrate these potential roles for the pro-regions of the a subunit have not been reported.

forms biologically

with FSH-suppressing

The nucleotide and derived amino acid sequences of aN are highly homologous among the human, pig, sheep, cow, and rat, particularly with respect to the number and location of four cysteine

pressed

gene ap-

pears to be regulated separately from that of the p genes, particularly in ovarian follicles at different stages of development, and the a gene is often expressed at higher levels than the P gene [for example, Engelhardt et al. (1993)l. Different immunoactive forms of the a subunit are present in follicular fluid of

of the sam-

ple prior to separation (Robertson et al. 1989), it is not known if aN exists in a free form. Cleavage of 58- to 32-kD inhibin, which removes aN, can occur extracellularly, for example in bovine plasma (McLachlan et al. 1986), but it is

Elsevier Science Inc., 1043-2760/95/$9.50

??

Endocrine Role of aN-Containing lnhibins

The 58-kD inhibin was the first form containing aN that was shown to have FSH-suppressing activity in vitro (Robertson et al. 1985). However, it remains to be determined whether this bioactivity results from cleavage to produce 32-kD inhibin. The nature of the biologically active form(s) of inhibin in plasma has been a contentious issue, with 32-kD inhibin originally being proposed as the major circulating form (McLachlan et al. 1986, Findlay et al. 199 1 ), principally on the basis that 58-kD inhibin was cleaved in plasma to the 32-kD form (McLachlan et al. 1986). However, larger forms of inhibin, which include aN, with in vitro biological activity have now been demonstrated in human plasma (Robertson et al. 1995). Indirect in vivo evidence for the FSH-suppressing activity of aN-containing inhibin(s) comes from experiments in which sheep were actively immunized against recombinant bovine aN (Russell et al. 1994a). These animals had increased concentrations of FSH

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305

0: lnhibin Subunit

is a required

/3,_B lnhibin Subunit Precursor

Pre Pro

aN

Precursor

aC

ProaN-&

0

Pro-aC

Pro p

lnhibin 6k

23k

p Monomer

Activin 20k

, 43k

I

a 105k B*B

-

’ Activin A,

-

P

ABSorB

95k

59k

a 31k I

fL

Figure 1. The subunit structures of inhibin, derivatives of the inhibin-a subunit, and activin.

(Figure 2) and reduced levels of inhibin (Figures 2 and 3) (measured by an aCsubunit-directed RL4) in the follicular and luteal phases of the estrous cycle, and increased numbers of corpora lutea (CL). The increased numbers of CL were thought to be a result of the increase in FSH concentrations stimulating folliculogenesis. It was suggested that the increased levels of FSH were due to reduced negative feedback by inhibin as a result of immunoneutralization by antibodies directed toward the aN portions of the higher molecular weight inhibins. This possibility was strengthened by the in vitro observations that an ovine antiserum against aN reduced both the immunological and biological potencies of 58-kD inhibin and bovine follicular fluid, both of which contain aN, but not that of 32-kD inhibin, which does not contain aN (Figure 4) (Russell et al. 1994b). It was postulated that the presence of aN antibodies could either prevent cleavage of aN that might be required to form biologically active inhibin (32 kD?) or interfere with the receptor/ligand interaction of inhibin containing aN. It is not known if cleavage of aN from larger forms of inhibin is a required event for expression of bio-

306

logical activity, and, to date, a specific receptor for inhibin has not been described that would allow testing of the second possibility. Latent precursors of the closely related growth factor, TGF-P, are not recognized by the TGF-P receptors and are thought to be important in regulating the autocrine and paracrine effects of TGF-P (Gentry et al. 1987, Wakefield et al. 1987). If removal of aN

event for the expression

of

biological activity, then activation of the enzyme(s) responsible for this cleavage represents an important point of regulation of inhibin bioactivity. Several proteases that may process inhibin precursors have been identified, including furin (Lindberg 1991) and an ovarianspecific protease (Hamabata et al. 1994). Heifers immunized against either of two peptides, each of I7 amino acids, based on 5’ (amino acids 8-20) and 3’ (153-I 67) sequences of bovine aN, respectively, also had increased numbers of CL, but the authors only observed a significant increase in FSH in the luteal phase of heifers that were immunized against the 3’ peptide (Morris et al. 1995). This same group of heifers also had higher concentrations of FSH (18.1 ng/mL) prior to the LH surge (- 16 to -5 h) compared to the controls (11.3 ng/mL), but the difference was not significant. In both the ewes (Russell et al. 1994a) and heifers (Morris et al. 1995), there were no significant effects of immunization against aN on either the time of onset or the amplitude of the preovulatory surges of LH and FSH or on the LH profiles (pulse frequency and amplitude) in these animals. The progesterone profiles of aN-immunized ewes were normal across the estrous cycle, although the mean concentrations tended to be higher than in controls, probably because of the increased number of apparent CL (Russell et al. 1994a). Direct measurements of estradiol concentrations have not been made in animals immunized against aN. However, indirect ev-

Figure 2. Mean t SEM peripheral concentrations of (a) FSH and (b) inhibin in daily plasma samples between day 2 and day 16 of the estrous cycle for control ewes (n = 41group) and three groups (3-5) of ewes immunized against bovine recombinant aN (n = 3-S/group). *p < 0.05 versus control. From Russell et al. (1994a), with permission.

3W

’Control ‘Group3 Group 4 ‘Group 5

01995, Elsevier Science Inc., 1043-2760/95/$9.50SSDI 1043-2760(95)00173-S

Control ‘Group 3 Group 4 Group 5

TEA4Vol. 6,No.9/10, 1995

immunoactivity after preincubabon with

10 /

-30

I

I

I

I

I

-20

-10

0

10

20

”

Time relative to LH surge (h) Figure 3. Mean f SEM inhibin concentration in peripheral plasma of control ewes (open circles, n = 7) and ewes immunized against bovine recombinant uN (closed triangles, n = 6) during the time of estrus. Time o represents the peak of LH and the dotted line, the onset of the LH surge. From Russell et al. (1994a), with permission.

idence suggests that estradiol production is likely to be within the normal range, because the aN-immunized ewes (Russell et al. 1994a) and heifers (Morris et al. 1995) showed normal estrous behavior and had normal, timely preovulatory surges of gonadotropins, both estrogen-dependent events. In summary, aN-containing inhibins, such as 58-kD and larger forms, exhibit FSH-suppressing activity in vitro, are present in the circulation, and most likely contribute to the feedback action on FSH secretion in vivo. Whether or not cleavage of the aN is necessary for inhibin to express its biological activity, or if there is some other role of aN such as extending the half-life of inhibin by acting as a carrier protein in the peripheral circulation, remains to be determined.

??

Paracrine

Role of OLN

Ewes immunized against recombinant bovine aN had impaired fertility, re-

flected in fewer lambs born per ewe mated (Findlay et al. 1989a). There were no apparent endocrine disturbances in these ewes that could explain the infertility (Findlay et al. 1989a, Russell et al. 1994a). As just described here, the animals had normal profiles of LH, raised levels of FSH, adequate preovulatory surges of gonadotropins at the appropriate time, and normal expression of estrus and cycle length. Subsequently, we showed that in ewes immunized against aN, the oocyte recovery rate (oocytes recovered from the oviducts 48 h after the time of the LH surge/number of CL) was significantly reduced (Table 1) (Findlay et al. 1989b and 1994, Russell et al. 1994a and 1995). This reduction in the recovery rate of oocytes in immunized ewes was observed in four independent experiments over several breeding seasons in different breeds of sheep and in the presence and absence of a low dose of equine chorionic gonadotropin to stimulate folliculogenesis.

32kDa ng/ml

58kDa ngiml

bFF IUlml

*significantly different from NI IgG treatedsample

Figure 4. h&bin immunoactivity and bioactivity of 32- and 58-kD bovine i&bin and bovine follicular fluid (bFF) after preincubation with antiaN IgG or preimnxune (NI) IgG. From Russell et al. (1994b), with permission.

The lower rates of oocyte recovery from the oviducts of aN-immunized ewes could have been due to the failure of follicles to ovulate. Indeed, some luteal structures had the appearance of luteinized unruptured follicles. Several of the ewes had large cystic follicles, no CL, and no oocytes recovered. Oocytes are released in some immunized ewes and do become fertilized in the oviducts (Findlay et al. 1994), indicating that loss

Table 1. Effect of immunization against (YN on the number of corpora lutea (CL) and oocyte recovery from the oviducts of ewes Number of CL Group

Number of ewes

Tofal

19 16 8 14

59 42 9 23

Control* Immunized* Control Immunized

Per ewe 3.1 rSD1.4 2.6 +-SD1.O 1.132SD0.13 1.65kSD0.23

Oocyfe recovery Total

%ofCL

References

45 7 8 9

76.3 16.7** 88.0 39.0**

Findlay et al. (1994) Russell et al. (1994a)

SD, standard deviation. *Treated with 800 IU eCG. “Significantly

below control 0, < 0.05)

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Figure 5. Representative Masson’s trichrome-stained sections of 2-day-old corpora lutea (CL) from a control ewe (a, X5 magnification; b, x20) and an aN-immunized ewe (c, X5; d, x20). Luteal tissue (L) in controls is confluent throughout the structure and contains deeply penetrating thecalbascular invaginations (T). The CL of the aN-immunized ewe contains a normal rupture stigma (R); however, luteal tissue is a thin layer of cells surrounding a large central antrum (A). Thecalbascular invaginations penetrate poorly and lack collagen (green) staining. From Russell et al. (1995). OThe Endocrine Society.

into the peritoneal cavity is unlikely. No oocytes were found in either the oviducts or uterine cavities of nine immunized ewes (Findlay et al. 1994), suggesting that egg transport is not accelerated. The data also showed that fertilization and subsequent egg development were normal in those ewes that did ovulate (Findlay et al. 1994).

308

It is our hypothesis that immunization of ewes against aN leads to a failure of the oocyte to be released from the follicle at the expected time of ovulation in a significant number of cases (Findlay et al. 1994, Russell et al. 1994a). Despite the fact that some of the immunized ewes had increased numbers of apparent CL (see earlier here), reduced

numbers of oocytes were recovered from the oviducts, and some of the CL-like structures had a cystic appearance. Therefore, we compared the morphology of the newly forming CL of control and aN-immunized ewes 2 days after the expected time of ovulation (Figure 5) (Russell et al. 1995). All nine CL from five control ewes had large rupture stigmata protruding through the ovarian surface. The luteal tissue was confluent within the CL structure, with many deeply penetrating, diffusely branched thecal/vascular invaginations. All eight CL from five immunized ewes had pronounced morphological differences compared with the controls. Although

01995, Elsevier Science Inc., 1043-2760/95/$9.50 SSDI 1043-2760(95)00173-5

TEM Vol. 6, No. 9/l 0, 1995

effective in reducing oocyte recovery and fertility in all animals. In sheep, we have noticed variation between experiments in the degree to which fertil-

been

a)

+ApMA

-ApMA Oh 12h -r--l--r--l-

24h

Oh

12h

Z4h

ity, oocyte recovery, and FSH concentrations were affected, which we attributed to small group sizes on some occasions

.__

a

L E _m

12~x3

d

loo0

and variation in antibody titers among individuals (Findlay et al. 1989a and b and 1994, Russell et al. 1994a and 1995). We observed that antibody titers in follicular fluid were approximately 50% of

Ir b.c

6

24

Hours

after

hCG

administration

6. Mean 2 SEM gelatinase activity in follicular fluid from two independent experiments (A and B) collected at 0, 12, or 24 h after hCG administration to control ewes (dark shaded bars) or crN-immunized ewes (light shaded bars). Numbers in bars equal the number of follicles tested. a, p < 0.05 versus zero control; b, p < 0.05 versus corresponding time control; c, p -C 0.05 versus olN at zero. From Russell et al. (1995). 0 The Endocrine Society. Figure

the rupture stigmata were normal, the center of each luteal structure was dominated by a fluid-filled antrum. The luteal tissue remained as a thin layer of cells surrounding the antrum, similar to granulosa cells lining the antrum of a preovulatory follicle. Thecal invaginations were rare, not branched, and failed to penetrate the luteal tissue. These CL contained relatively little staining for interstitial collagen in the thecal areas compared with CL of control ewes. Oocyte recovery was 58% in immunized ewes and 88% in controls. Despite these abnormalities in CL structure, earlier studies had shown that luteal function with respect to progesterone concentration and profile was normal in immunized ewes (Russell et al. 1994a). These effects of immunization against IXN on CL morphology 2 days after the expected time of ovulation are consistent with incomplete tissue remodeling during ovulation and CL formation. Although this did not affect luteal function, it may account for the reduced recovery of oocytes in these animals, because oocyte release could have been impaired. Immunization against CXN has not

TEM Vol. 6, No. 9110, 1995

those in peripheral plasma (Findlay et al. 1994), so in some ewes, antibody levels may have been inadequate to influence olN within the preovulatory follicle, but still be sufficient to immunoneutralize CXNin the peripheral circulation. In addition, there may be variation among sheep in the epitopes recognized by the antisera raised by active immunization. Morris et al. (1995) did not observe any significant effects of immunization of heifers against the 5’ and 3’ peptides of bovine aN on conception or calving rates. It is difficult to compare directly the heifer and the sheep studies, because different antigens were used and the methods of measuring and expressing titers are different. Both species had increased CL numbers as a result of immunization, and this was more clearly related to an increase in FSH in ewes than in heifers. Evidence for ovulation in the heifer study was based on the presence of normal luteal structures with little or no fluid-filled lacunae observed by transrectal ovarian ultrasonography. This suggests that the aN-immunization protocol for the heifers did not grossly alter CL structure as it did in the sheep. However, this conclusion needs confirmation by a histological examination of CL in uN-immunized heifers.

??

Mechanisms of Ovarian Action of CXN

During ovulation, the extracellular matrix of the follicle and ovarian wall is degraded at the follicular apex, weakening its tensile strength to allow rupture of the follicle and extrusion of the oocyte. The importance of digestion of interstitial collagen and modifications to the components of the basement membranes in this remodeling process at the time of ovulation is well documented [see Russell et al. (1995)], and involves both interstitial collagenase and gelati-

01995, Elsevier Science Inc., 1043-2760/95/$9.50

SSDI

Oh

12h

24h

Figure 7. (a) Gelatin zymography of pools of follicular fluid collected from control (C) or uN-immunized (I) ewes 0, 12, or 24 h after hCG. (b) Image analysis quantification of the total areas digested by active and latent MMP-2 in samples without APMA activation. (c) Relative proportion of active and latent MMP-2 species in ovine follicular fluid samples without APMA treatment. From Russell et al. (1995). 0 The Endocrine Society.

nases, all members of the matrix metalloproteinase (MMP) family of enzymes. We had shown that immunization of ewes against aN resulted in altered morphology of the newly forming CL, in particular, an apparent lack of remodeling of the basement membrane of the follicle to allow invagination of the the&vascular layers (Russell et al. 1995). The MMP that target type IV collagen found in basement membranes are MMP-2 (72~kD gelatinase A), MMP-9 (92-kD gelatinase B), and MMP-3 (stromelysin-1, which has not been identified in ovarian tissue). Therefore, we examined the effect of immunization against aN on the activity and type of gelatinases in preovulatory sheep follicles collected at the time of the LH(hCG) surge (time zero), and at + 12 and +24 h (the expected time of ovulation in the ewe) (Russell et al. 1995). It was found that in control ewes, gelatin-digesting activity in follicular fluid

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309

lar fluid and in recombinant i&bin-o is not yet known. The evidence suggested that the forms of inhibin-cx associated with this activity include a 57-k~ precursor, as well as smaller processed forms. The relative potencies of these molecules were of the order of SO- to lOO-fold lower than that

Pituitary [

of pituitary FSH, but the authors concluded that because it&bin-or precursors OCCLXin follicular fluid at concentrations exceeding 2.5 pg/mL, they could still exert a physiological role as modulators of FSH action. There is no obvious relationship between the putative FSH binding activity of the i&bin-o subunit components and the effects of active immunization against CXNon FSH levels, ovulation, and gelatinase activity.

Ovariar1 Follicle

??

Digestion of Collagen types I and IV

1 Thinning and rupture of follicle wall Figure 8. A hypothesis of the sites of action of aN on the ovulatory cascade induced by the LH surge. __ increased nearly twofold by +24 h, whereas in oN- immunized ewes the activity up to +24 h was the same as the level at 0 h (Figure 6). Gelatin zymography of follicular fluid revealed bands of gelatinase of 72/67 kD, consistent with latent and active MMP-2; the area of substrate digested by latent and active MMP-2 increased with approaching ovulation in follicles of control ewes and was decreased by CXN immunization (Figure 7). These data suggest that MMP-2 has a role in the tissue remodeling processes of ovulation and CL formation in the ewe, and that immunization against aN, which can reduce fertility in ewes, affects the preovulatory cascade of intrafollicular proteolytic activity, reducing MMP-2 levels and disrupting normal CL formation (Russell et al. 1995). Further studies are in progress to define the cellular origins of these MMP and their inhibitors, and to understand how they may be influenced by orN. Particular points of interest are the relationship between the expression of aN in follicles before and after the LH surge, and the effects of olN on ovulation, CL formation, and gelatinase activity. Before the LH surge, expression of

310

the a subunit in granulosa cells of the preovulatory sheep follicle is high (Engelhardt et al. 1993) and secretion of immunoactive inhibin by the ovary is relatively high (Baird et al. 199 1, Russell et al. 1994a). After the LH surge, there is a precipitous decline in the expression of the (Ysubunit in the preovulatory follicle, accompanied by a decline in the inhibin levels in plasma. Similar observations have been made on the expression of inhibin-cy by luteinizing bovine granulosa cells in serum-free conditions (Luck et al. 1990). This implies that either the effects of aN on the preovulatory cascade are set in place prior to induction of the ovulatory cascade by the LH surge, or that there is sufficient olN present in the ovulatory follicle after the LH surge to influence the cascade. Another potential action of olN in the ovary concerns autocrine or paracrine modulation of FSH action (Schneyer et al. 1991). Proteins derived from the inhibin-cy subunit were shown to have the capacity to inhibit binding of FSH to its receptor. The exact nature of the inhibin-a precursor molecule(s) responsible for this FSH receptor binding activity identified in human and porcine follicu-

01995, Elsevier Science Inc., 1043-2760/95/$9.50

Future

Studies

The strategy employing active immunization to investigate the role of aN in fertility does not discriminate the form of the endogenous active peptide involved in the observed effects on ovulation. Hence, although it can be concluded that aN has an important role in fertility regulation in the ewe, it remains to be determined whether this activity is a function of the discrete aN peptide or of other forms of the inhibin dimer or free OLsubunit that contain N. Further elucidation of the biologically relevant forms of the a subunit and inhibin is needed. Ligand binding studies are also required to ascertain if there are specific high-affinity receptors for aN-related peptides, as well as to confirm whether or not such peptides function through competition with FSH. The potential for processing of the larger molecular weight a subunit during testing in these biological systems is a possible confounding issue. In order to fully confirm the action of immunization against aN on ovulation, it will be necessary to demonstrate that oocytes are retained within the unruptured follicles, and that the time course of ovulation has not been interrupted. The mechanism(s) by which immunization has interrupted ovulation is not clear and could involve any of several events in the biochemical cascade that leads to ovulation (Figure 8) (Lipner 1988). Current opinion is that prostaglandins and plasminogen activator have roles in the regulation and activation of MMP (Lipner 1988) in which

SSDI 1043-2760(95)00173-5

TEM Vol.6,No. g/IO,1995

case it can be postulated that aN could act at’one or more sites in the cascade (Figure 8). Although MMP-2 production was shown to be mainly by the thecal compartment in the rat (Hurwitz et al. I993), it could also come from leukocytes, which are an important component of the ovulatory process (Brannstrom and Norman, 1993). The site(s) of MMP-2 production in the ovary, its relationship to leukocyte infiltration, and the effects of aN on these parameters and the integrity of the basement membrane of follicles deserve further study. A limitation to studying the potential actions of aN has been the lack of sufficient pure native or recombinant protein. Native material is a relatively minor component of the purification of inhibin from follicular fluid (Robertson et al. 1989). Recombinant bovine aN is a fusion protein (Findlay et al. 1989a) that includes a small amino acid extension derived from the galactosidase protein and lacks glycosylation, so it would not be folded like the native protein, making it less desirable for definitive studies. Expression of aN in mammalian cells is being explored. Overall, the evidence summarized in this review suggests a unique physiological significance for the precursor peptides of the inhibin-a subunit in both the endocrine and paracrine control of fertility.

subunits and ovarian secretion of i&bin and estradiol at various stages of the sheep estrous cycle. Biol Reprod 49:281-294.

cleic acid for inhibin

Findlay JK, Tsonis C, Doughton B, et al.: 1989a. Immunization against the aminoterminal peptide (N) of the alpha43 subunit of inhibin impairs fertility in sheep. Endocrinology 124~3122-3124. Findlay JK, Tsonis C, Doughton B. et al.: 198%. The amino-terminal peptide (aN) of the alpha 43 kD subunit of inhibin (~43) influences fertility in sheep [abstl. Proc Aust Sot Reprod Biol 21:134. Findlay JK, Clarke IJ, Luck MR. et aI: 1991 Peripheral and intragonadal actions of inhibin-related peptides. J Reprod Fert SuppI 43:139-150. Findlay JK, Russell

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia, The Buckland Foundation, and Biotech Australia Pty Limited.

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