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Summary. Graafian follicles from rats treated for 1 or 2 days with pentobarbitone sodium (Nembutal) were similar in appearance to pro-oestrous preovulatory follicles, but after 3 or 4 days of treatment early atretic changes were recognized. Ovulatory efficiency decreased to 88, 70, 52 and 31% after 1, 2, 3 and 4 days of treatment, respectively. The mean ± s.e.m. rate of accumulation (ng/follicle/24 h) of progesterone, androstenedione and oestradiol was 3·6 ± 0·7, 4·0 ± 0·3 and 18·9 ± 3·9 respectively in preovulatory follicles and 10·2 + 1·7, 0·9 ± 0·1 and 1·9 ± 0·4 respectively in follicles explanted from rats treated for 4 days with Nembutal. Addition of LH (5 μg/ml) to the culture medium stimulated steroid accumulation by both types of follicles. Thus atretic follicles are characterized by impaired androgen and oestradiol formation. Addition of testosterone (1 μg/ml) to the culture medium increased the accumulation of oestradiol by atretic follicles. It is inferred that the early stages of atresia of rat follicles are distinguished by a deficiency in the activity of enzymes responsible for the conversion of progesterone to androgens that can serve as substrates for aromatization.

Summary.

Follicular oocytes were examined at various times preceding ovulation. The timing of the preovulatory changes was established in relation to the `critical period' and to ovulation time. The earliest detectable change in the oocyte, loss of the germinal vesicle and the nucleolus, occurred about 2 hr after the presumed time of lh release, or about 9 hr before ovulation. The first polar body was extruded about 4 hr before ovulation. The morphology of the dictyate oocyte as revealed by interference contrast microscopy is described.

Nalbandov (1972) suggested that the ovum produces a "luteostatic" substance which prevents luteinization of granulosa cells. This suggestion was derived from three lines of observation: (1) surgical ovectomy in vivo induced luteinization and increased the output of progesterone into the ovarian venous blood of rabbits (El-Fouly, Cook, Nekola & Nalbandov, 1970) and pigs (Nalbandov, 1972); (2) rat granulosa cell monolayers cultured with only a few oocytes luteinized, whereas those cultured in the presence of many ova retained their granulosa cell morphology (Nekola & Nalbandov, 1971); and (3) oocytic degeneration on the 3rd day of culture of rat follicles coincided with morphological luteinization of the mural granulosa (Stoklosowa & Nalbandov, 1972).

Department of Physiology, University of Maryland, School of Medicine, 660 West Redwood Street, Baltimore, Maryland 21201, U.S.A.

(Received 16th September 1974)

The original finding of Pincus & Enzmann (1935) that isolated rabbit oocytes will mature spontaneously in vitro has been extended to nine other mammalian species (Donahue, 1972), including the pig (Edwards, 1965; Foote & Thibault, 1969; McGaughey & Polge, 1971). The main objectives of the present communication are (a) to describe a culture system for studying the maturation of pig oocytes which can also be used for other ovarian cell types, and (b) to compare the ability of oocytes isolated from follicles differing in their developmental stage to undergo maturation.

Oocytes were collected from pig ovaries obtained at a local slaughterhouse. Within 20 to 30 min after the pigs had been killed the ovaries were excised and placed in 0·9% NaCl plus 100 i.u. penicillin/ml and 100 μg streptomycin/ml

Summary. Administration of PMSG to 26-day-old rats did not increase the total number (non-atretic and atretic) of preantral (Type 4) and antral (Types 5 and 6) follicles but changed the proportion between non-atretic and atretic follicles. By 12 h after PMSG administration only 7% of Type 5 follicles were atretic, and no atretic follicles of Type 6 were observed, as compared to 52 and 62% atretic follicles, respectively, in the saline-treated controls. PMSG did not decrease the percentage of atresia in preantral (Type 4) follicles. The treatment was associated with a sharp fall by 12 h in the pyknotic index of antral (Types 5 and 6) follicles. It is concluded that PMSG causes superovulation in the rat by rescuing follicles of Types 5 and 6 from atresia and allowing them to reach ovulation.

Meiotic maturation of mammalian oocytes is a protracted process, subject to multiple stop-go controls (Text-fig. 1). The meiotic process is initiated during fetal life and is arrested shortly after birth at the stage of diplotene. Normally meiosis is resumed in adult life following the preovulatory surge of gonadotrophins (Ayalon, Tsafriri, Lindner, Cordova & Harell, 1972; Vermeiden & Zeilmaker, 1974; Tsafriri et al., 1976a; Dekel, Hillensjö & Kraicer, 1979). Preovulatory resumption of meiosis includes the breakdown of the germinal vesicle (GVB), expulsion of the first polar body and the progress to the metaphase of the second meiotic division. Nevertheless, since GVB is the first change occurring and is widely used as an endpoint for assessing the resumption of meiosis, we shall refer to GVB as 'resumption of meiosis' or as 'oocyte maturation'.

Summary.

Isolated Graafian follicles cultured intact were used as a test-system for the meiosis-inducing action of hormonal preparations and for analysing the mediation of this hormone effect.

When enlarged follicles were explanted from rats on the day of pro-oestrus before 14.00 hours, i.e. before the preovulatory lh-surge, the oocytes remained in the dictyate state of meiosis throughout an 18-hr culture period. Completion of the first meiotic division could be induced by addition of lh or prostaglandin E₂ to the culture medium, or by microinjection of dibutyryl cyclic AMP into the antrum of the cultured follicle. Addition of hcg or fsh to the culture medium was also effective, though possibly due to lh-like activity present in these preparations. Prostaglandin F_2α, at 2·8 × 10⁻⁵ m, was only partly effective; prolactin, progesterone, 20α-dihydroprogesterone, oestradiol-17β, linolenic acid and adenosine-5′-monophosphate were completely ineffective. The maturation-inducing action of lh was not blocked by cyanoketone, an inhibitor of steroid synthesis.

Addition of lh to the culture medium stimulated the formation of cyclic AMP by the isolated follicles. Exogenous cyclic AMP enhanced protein kinase activity in the supernatant fraction of follicular homogenates.

It is proposed that the action of lh on oocyte maturation involves the mediation of the adenyl cyclase/cyclic AMP system and possibly of the prostaglandins. An action of steroids could not thus far be implicated. The experimental model described permits study of the mechanism of the meiosis-inducing action of lh under controlled conditions in vitro.

Summary. Oxygen consumption was measured in denuded oocytes and oocyte–cumulus complexes isolated from atretic rat follicles. Adult cyclic rats or immature PMSGtreated rats were used, and follicular atresia was induced by hypophysectomy on the morning of pro-oestrus or by repeated pentobarbitone injections beginning on the day of pro-oestrus. The later stages of atresia were accompanied by meiosis-like changes in the oocytes. Oxygen consumption by oocytes that had resumed meiosis (germinal vesicle breakdown, GVB) was higher than in oocytes with an intact germinal vesicle, a change similar to that observed in oocytes maturing in healthy follicles. This may indicate that the meiotic process in the atretic follicles is similar to that in normal ones.

Oxygen consumption by the cumulus cells was not altered during pentobarbitoneinduced atresia. Hypophysectomy led to a rapid and marked increase in cumulus oxygen consumption in cyclic rats but there was no change in PMSG-treated young animals. Since both pentobarbitone-treatment and hypophysectomy result in follicular atresia, but changes in cumulus respiration occurred only in hypophysectomized adult rats, it is concluded that an increase in cumulus respiration is not inherent to the atretic process.

Summary.

Serum levels of lh, fsh and prolactin were determined in pro-oestrous rats by radioimmunoassay, and the time of commitment of oocytes to undergo maturation was defined by explanting follicles at hourly intervals into a hormone-free medium. All three pituitary hormones showed a rise during the afternoon of pro-oestrus. The prolactin level was still elevated on the morning of oestrus, and serum fsh showed a secondary rise at that time. Administration of pentobarbitone-sodium at 14.00 hours abolished the afternoon rise in the levels of lh and fsh, but not of prolactin, and prevented the induction of oocyte maturation.

The percentage of oocytes that completed the first meiotic division when explanted within intact follicles increased during the afternoon of pro-oestrus from <5% at 14.30 to 80% at 17.30 hours. This maturation commitment preceded the dissolution in vivo of the germinal vesicle by about 1½ hr. Its attainment closely followed the rising curve of serum lh concentration, indicating that only brief exposure of the follicle to lh is required to initiate ovum maturation.

Summary. (1) Follicles vary greatly in their response to a hormonal stimulus. Some of the factors that influence the sensitivity of the rat follicle to ovulatory hormones are discussed in this review. (2) The responsiveness of the rat ovary to gonadotrophins undergoes striking augmentation during the 2nd week of postnatal development with respect to cyclic AMP formation, cAMP-stimulated protein kinase activity, inducibility of ornithine decarboxylase and oestrogen secretion. (3) FSH, probably in concert with oestrogen, sensitizes the follicle to subsequent stimulation of adenylyl cyclase by LH. This synergism, based on heterologous receptor induction, operates during prepubertal as well as cyclic follicular maturation. (4) The ovarian LH-stimulated adenylyl cyclase possesses a guanine nucleotide regulatory site. Occupation of this site by GTP or Gpp(NH)p reduces the concentration of LH required for half-maximal stimulation of the enzyme and increases its activity (V_max). (5) Androgen synergizes FSH in stimulating progestin secretion by cultured immature granulosa cells, though the steroid is known to antagonize the FSH action on granulosa cell proliferation. Thecal androgen may be required for the maturation of the FSH response mechanism in granulosa cells of preantral follicles, while promoting atresia in large antral follicles. (6) Continued exposure of the follicle to high concentrations of LH, FSH, or PGE-2 results in refractoriness of adenylyl cyclase to further stimulation by the same hormone. Desensitization may be transient (PGE-2) or protracted (LH) and seems to depend on the synthesis of a macromolecular inhibitor that affects the coupling between hormone receptor and adenylyl cyclase. This mechanism may account for the shut-down of follicular steroid production at ovulation, which depends on cAMP, and permit the expression of processes that are inhibited by cAMP. (7) Granulosa cells exert a restraining influence on oocyte maturation which may be transmitted by direct contact and perhaps by substances released into the follicular fluid; this inhibition is reversed by LH. The sensitivity of the oocyte to the meiosis-triggering action of LH increases, and its susceptibility to inhibition by follicular fluid extracts decreases, during the final stages of preovulatory development. (8) Specific binding sites for LH are restricted to the theca interna and the peripheral layers of the membrana granulosa of the preovulatory follicle. Extensive gap junctions between granulosa cells provide an anatomical basis for the propagation of the signal generated by LH to the interior of the follicle. The presence of such gap junctions between cytoplasmic extensions of the coronal cells and the oocyte may serve to control the membrane potential of the oocyte by electrical coupling. Depolarization of this membrane precedes ovum maturation in non-mam-malian species. (9) Prostaglandin synthesis within the follicle is stimulated by LH and is essential for LH-induced follicular rupture. Cells of the external theca contain abundant microfilaments and are rich in actin and smooth-muscle myosin, demonstrable by immunofluorescence. These smooth muscle-like cells may be a target for prostaglandins and/or adrenergic neurotransmitters during ovulation and assist extrusion of the cumulus mass. (10) Actin and myosin occur beneath the oolemma of the mammalian oocyte and may play a part in the dynamics of the maturation divisions. (11) Thecal and granulosa cells in ovarian slices incorporate ³⁵SO₄ into chondroitin sulphates and heparin-like substances and rapidly secrete these into the antral fluid. LH suppresses the synthesis of these sulphated glycosaminoglycans; progesterone probably mediates this inhibition. Since the antral S-GAGs include heparin, this secretion may have regulatory actions within the follicle beyond the hydrodynamic effects postulated earlier. (12) Delayed effects of hormones on the sensitivity of the follicle to the same hormone or to the action of a heterologous hormone, and the successive appearance and redistribution of different hormone receptors and specialized membrane components may be significant elements in the programming of a precisely timed developmental sequence that is a characteristic feature of the life history of the follicle.