Potential local regulatory functions of inhibins, activins and follistatin in the ovary

The changing pattern of granulosa cell expression of inhibin/activin subunits and follistatin during follicle development and their differential regulation by extrinsic and intraovarian factors supports evidence from functional studies, mostly in vitro, that these proteins have important roles in folliculogenesis, oocyte maturation and corpus luteum function. Gonadal inhibins function as negative feedback hormones to regulate the synthesis and secretion of pituitary FSH, a key determinant of follicle development, but there is little supportive evidence for a peripheral endocrine role for ovary-derived activins or follistatin in this regard. However, activins and follistatin are expressed in numerous other tissues, including anterior pituitary, and they are firmly implicated as local intrapituitary regulators of FSH secretion. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation and upregulation of FSH receptors, P450arom, oestrogen synthesis, granulosa cell LH receptors and enhancement of oocyte maturation. Through its activin-binding role, follistatin can reverse each of these activin-induced responses. In addition to their endocrine feedback role, granulosa-derived inhibins can sensitize theca cells to LH, thereby enhancing the production of androgens, an essential requirement for follicular oestrogen synthesis. Activins can oppose this effect and suppress thecal androgen production. Granulosa cells overproduce inhibin a subunit precursor relative to betaA/betaB subunit precursors and evidence indicates that different parts of the inhibin a subunit precursor have intrinsic biological activities distinct from inhibin alphabetaA/B dimer, and serve as additional local modulators of follicle and corpus luteum function.     

Differential effects of activin A on basal and gonadotrophin-induced secretion of inhibin A and progesterone by granulosa cells from preovulatory (F1-F3) chicken follicles

Previous work has shown that activin A is expressed selectively within the theca rather than the granulosa layer of preovulatory chicken follicles. In the present study, this finding was verified and the potential paracrine actions of activin A on basal and gonadotrophin-induced secretion of inhibin A, inhibin B and progesterone by granulosa cells from the three largest preovulatory follicles (F1-F3) were investigated. Treatment with activin A (0, 0.25, 2.5 and 25 ng ml(-1)) alone increased inhibin A secretion markedly in a follicle- and time-dependent manner, with the greatest response (up to 15-fold increase; P < 0.0001) in F1 follicles after 3 days of treatment. In contrast, activin A alone had no effect on progesterone output at any time. Cells from F3 follicles were more responsive to FSH than were F1 cells in terms of both inhibin A (P < 0.02) and progesterone (P < 0.01) secretion. Furthermore, activin A greatly enhanced FSH-induced secretion of both inhibin A (up to tenfold; P < 0.0001) and progesterone (up to sixfold; P < 0.0001). In terms of LH-induced inhibin A and progesterone secretion, cells from F1, F2 and F3 follicles showed similar responses. Co-treatment with activin A enhanced LH-induced secretion of inhibin A markedly (up to ninefold; P < 0.0001) but had only a marginal effect on LH-induced progesterone secretion (up to twofold; P < 0.001). The presence of activin receptor subtypes IA, IB, IIA and IIB in cultured granulosa cells from F1, F2 and F3 follicles was demonstrated using immunocytochemistry. These findings support the hypothesis that activin A secreted by the theca layers of avian preovulatory follicles exerts a local paracrine action on granulosa cells to modulate 'basal' inhibin A secretion and to upregulate gonadotrophin-induced secretion of both inhibin A and progesterone. However, the extent to which this local role of activin A contributes to the generation of the preovulatory LH-progesterone surge remains to be established.     

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta ) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, BMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. In addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.     

Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season

The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.     

The effects of FSH and activin A on follicle development in vitro

Numerous studies have reported on the roles of activins in gonadal regulation; however, little is known about their specific roles in early folliculogenesis. Ovarian follicular growth was investigated in 10-day cultures of day 4 postnatal whole ovaries treated with activin A (ActA; 50?ng/ml), with or without FSH (100?ng/ml) in vitro. We hypothesized that treatment with ActA±FSH would affect rates of growth and atresia in follicles. None of the treatments affected primordial follicle activation, and antral follicles were not observed after 10 days in culture. Primordial follicle numbers from all treatment groups were ～20% of those in day 4 fresh ovaries, indicating that activation had occurred. In the presence of ActA, preantral follicle numbers increased significantly (P<0.0001). ActA alone decreased the proportion of atretic follicles in the primary and preantral classes, whereas the combined treatment of ActA+FSH increased the proportion of atretic preantral oocytes. Real-time PCR analysis revealed that follistatin, FSH receptor, and activin βA and βB subunits were all expressed at significantly higher levels in the ActA-only treated group but not in the ActA+FSH group. Here, we report novel findings supporting the role of FSH in primordial follicle survival through an action on apoptosis and a stimulatory role of ActA in the primordial to primary and preantral stages of follicle development, suggesting an inhibitory action of activin on oocyte apoptosis.     

Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression

The LH surge initiates the luteinization of preovulatory follicles and causes hormonal and structural changes that ultimately lead to ovulation and the formation of corpora lutea. The objective of the study was to examine gene expression in ovarian follicles (n = 11) collected from pigs (Sus scrofa domestica) approaching estrus (estrogenic preovulatory follicle; n = 6 follicles from two sows) and in ovarian follicles collected from pigs on the second day of estrus (preovulatory follicles that were luteinized but had not ovulated; n = 5 follicles from two sows). The follicular status within each follicle was confirmed by follicular fluid analyses of estradiol and progesterone ratios. Microarrays were made from expressed sequence tags that were isolated from cDNA libraries of porcine ovary. Gene expression was measured by hybridization of fluorescently labeled cDNA (preovulatory estrogenic or -luteinized) to the microarray. Microarray analyses detected 107 and 43 genes whose expression was decreased or increased (respectively) during the transition from preovulatory estrogenic to -luteinized (P<0.01). Cells within preovulatory estrogenic follicles had a gene-expression profile of proliferative and metabolically active cells that were responding to oxidative stress. Cells within preovulatory luteinized follicles had a gene-expression profile of nonproliferative and migratory cells with angiogenic properties. Approximately, 40% of the discovered genes had unknown function.     

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice

Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin beta(C), increased. Inhibin/activin beta(A) and beta(B) subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the beta(A) subunit also detected in peritubular myoid cells. The alpha, beta(A), beta(B) and beta(C) subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While beta(A) and beta(B) subunit proteins were observed in spermatogonia and spermatocytes in immature testes, beta(C) was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear beta(A) subunit protein was observed in primary spermatocytes and nuclear beta(C) subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.     

Differences in follicular function of 3-month-old calves and mature cows

After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Müllerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Müllerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation.     

Expression and localization of inhibin alpha,inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.     

Preovulatory follicle characteristics and oocyte competence in repeat breeder dairy cows

The varied and elusive etiology of repeat breeding (RB) in dairy cows necessitates evaluation of oocytes and follicles, which have not previously been assessed together. Accordingly, we evaluated characteristics of preovulatory follicles and the competence of oocytes in control (CTL) and RB cows. The estrous cycles of 35 cows (18 CTL and 17 RB) were synchronized using PGF_2α and estrus detection. Cows with a corpus luteum were treated with PGF_2α and, 14 to 15 d after a visible behavioral estrus, they were administered a second PGF_2α, followed 48 h later by follicular fluid (FF) aspiration of the preovulatory follicles. Estradiol (E₂)-active preovulatory follicles did not differ in diameter between the 2 groups of cows. However, FF of RB cows had higher E₂ concentrations than that of CTL cows: 1,854.9 and 1,073.6 ng/mL, respectively, but similar androstenedione and progesterone concentrations. In the second part of the study, 14 consecutive ovum pick-ups (OPU) were performed in 5 CTL and 5 RB cows, at 3- to 4-d intervals. The RB and CTL cows did not differ in average numbers of follicles available per cow per session (7.1 and 7.3, respectively), oocyte recovery rates (42.2 and 44.1%, respectively), or cleavage rates (57.6 and 63.4%, respectively), but blastocyst production was markedly less in RB than in CTL cows (12.5 and 29.2%, respectively). We conclude that part of the RB cows' etiology occurs at an earlier phase of folliculogenesis, thereby impairing oocyte competence, and subsequently reducing the probability of normal fertilization, which diminish embryo vitality and development.     

Effects of high dietary crude protein on the characteristics of preovulatory follicles in dairy heifers

The objectives were to examine the effect of high dietary crude protein on characteristics of preovulatory follicles in dairy heifers. Eight Israeli-Holstein heifers, 4 fitted with rumen fistula and 4 intact, were assigned to 1 of 3 treatments in a replicated (n = 2) 4 × 3 incomplete Latin square design with 39-d periods. Treatments were: low (6.0%; LP), moderate (13.0%; MP), and high (20.0%; HP) crude-protein diets, containing 1.27 Mcal NE_L/kg dry matter. Diets were based on approximately 66% wheat straw and various proportions of ground corn grain and soybean meal. The estrous cycles of the heifers were synchronized, and 14 d after behavioral estrus, heifers received PGF_2α injections. After a further 40 h, at d 39 of each period, follicular fluid (FF) was aspirated from follicles of diameter >7 mm. The intake of the LP diet was 9% lower than that of MP and HP diets. Rumen ammonia and plasma urea nitrogen concentrations were highest in the HP and lowest in the LP, with intermediate levels in MP diets. No differences were found between treatments in plasma and FF concentrations of glucose and nonesterified fatty acids. High-protein diets increased urea concentrations very similarly in preovulatory FF and in plasma. No differences were observed between diets, in preovulatory follicle diameters and concentrations of androstenedione. However, higher estradiol and progesterone concentrations in FF were observed under the HP than under the MP diet, with no difference between diets in estrogen to progesterone ratio. It can be concluded that high concentrations of urea in plasma, caused by high dietary crude protein, penetrated into preovulatory follicles, but did not impair preovulatory characteristics. This lack of detrimental effects might be attributed to the use in this study of nonlactating heifers, which have fewer nutritional and physiological constraints and eliminate negative effects of potential interactions with high urea on dairy cows’ reproductive systems.     

Induction of birth in the bandicoot (Isoodon macrourus) with prostaglandin and oxytocin

The aim of this study was to compare the actions and interactions of gonadotrophins (LH and FSH) and an analogue of insulin-like growth factor I (LR3-IGF-I) on the secretion of inhibin A, inhibin B and progesterone by cultured chicken granulosa cells derived from the three largest (F1--F3) follicles of the preovulatory hierarchy. Treatment with LH or FSH promoted marked dose-(P < 0.0001) and time- (P < 0.0001) dependent increases in both inhibin A and progesterone secretion, with the magnitude of response (< 15-fold compared with basal) increasing over time in culture. Concentrations of inhibin B were below the detection limit in all samples. Initially, F1 cells were more LH-responsive than were F3 cells in terms of progesterone secretion (P < 0.02) but this difference between follicles decreased over time in culture. In contrast, LH-induced inhibin A secretion tended to be highest from F3 cells, although this was not significant. Cells from F3 follicles were consistently more FSH-responsive than F1 cells in terms of both progesterone (P < 0.01) and inhibin A (P < 0.02) secretion. Initially, F1 cells were more responsive to LR3-IGF-I than were F3 cells in terms of progesterone secretion (P < 0.001) but were less responsive in terms of inhibin A secretion (P < 0.001). Again, these inter-follicle differences decreased over time in culture (not significant on day 3 of treatment). Co-treatment experiments showed that LR3-IGF-I enhanced both LH- and FSH-induced secretion of inhibin A and progesterone in a time- (P < 0.001) and follicle- (P < 0.001) dependent way. Initially, F1 cells showed highest LR3-IGF-I enhancement of LH-induced inhibin A and progesterone secretion; in contrast, F3 cells showed the highest LR3- IGF-1 enhancement of FSH-induced inhibin A and progesterone secretion. These inter-follicle differences persisted over time in the case of FSH-induced hormone responses but not in the case of LH-induced responses, even though the relative degree of LR3-IGF-I enhancement increased markedly over time. Collectively, these data support a positive role for IGF-I, presumably of thecal origin, as an amplifier of gonadotrophin action on granulosa cell inhibin A and progesterone production by preovulatory chicken follicles.     

Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6-8?mm) follicles (6-8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1-8?mm) follicles compared with selected (≥9?mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6-8?GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21?h preculture with rhBMP6 followed by a 3?h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6-8?GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8?mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.     

Effect of number and diameter of follicles on plasma concentrations of inhibin and FSH in mares

The role of the number of follicles and circulating immunoreactive inhibin in the decrease in plasma FSH concentrations that occurs during development of a follicular wave was studied in mares. All follicles > or = 6 mm in diameter were ablated by ultrasound-guided transvaginal aspiration of follicular fluid on day 10 after ovulation. During the subsequent wave, all follicles, the three largest follicles (three follicle group), the largest follicle (single follicle group) or no follicles were retained and the remaining follicles were ablated before they reached > 10 mm in diameter (n = 10-11 mares per group). Ablation of new follicles was continued until the day on which the largest follicle of the new wave reached 25 mm in diameter (day 18 after ovulation in the 'no follicle' group). Diameters of retained follicles were measured once a day by transrectal ultrasonography. Plasma samples were taken once a day and analysed by radioimmunoassay for concentrations of FSH and immunoreactive inhibin (includes dimeric inhibin as well as free alpha-subunit forms). Data were normalized to the day of the expected start of the decrease in plasma FSH concentrations (day 0: largest follicle 13 mm in diameter in the follicle-retained groups). A simultaneous increase in circulating concentrations of FSH (P < 0.05) and immunoreactive inhibin (P < 0.05) occurred before the largest follicle reached 13 mm in diameter, which indicates that immunoreactive inhibin produced by follicles < 13 mm in diameter did not suppress FSH. Plasma concentrations of FSH decreased (P < 0.05) and immunoreactive inhibin concentrations increased (P < 0.05) after day 0 in the follicle-retained groups. A slower decrease in FSH concentrations was associated temporally with a delay in the increase in immunoreactive inhibin concentrations in the 'single follicle' group relative to the 'three follicle' and 'all follicle' groups. All follicle-retained groups had similar plasma concentrations of FSH and immunoreactive inhibin after the expected beginning of deviation in growth rates between the two largest follicles (largest follicle 22-23 mm in diameter). These results indicated that the decrease in plasma FSH concentrations from the start of the decrease until the expected day of deviation was a function of multiple follicles of a wave and was attributable to the secretion of inhibin. Thereafter, the largest follicle alone accounted for the continued FSH suppression.     

Gene expression for LH receptor, 17 alpha-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17 alpha-hydroxylase (17 alpha OH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n = 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17 alpha OH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17 alpha OH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR. In summary, our study suggests that in spite of the presence of 17 alpha OH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.     

Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.