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MARGARET RYLE

Summary.

Ovaries of infantile mice were cultured in vitro with no hormone, lh or fsh for 2 or 5 days, [3H] thymidine being present in the medium for the last 24 hr of culture. Each ovary was serially sectioned and autoradiographs were prepared. The effects of the treatments on follicle numbers and on the extent to which they were labelled were examined in groups of follicles of different sizes. Growth was stimulated by fsh in follicles of all sizes both on Day 2 and Day 5, but lh did not stimulate growth in primordial follicles and had relatively little effect on one- and two-cell layer follicles. It greatly accelerated growth in some with three- and four-cell layers on Day 5, but not on Day 2. It also appeared to stimulate mitosis in the theca interna whereas fsh appeared to diminish it. A few follicles grew rapidly on Day 5 in the absence of any hormone. Spindle formation in oocytes was associated with rapid follicle growth but not with hormone treatment. Possible explanations of the delayed effect of lh are discussed.

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J. J. Peluso, S. Luttmer and M. L. Gruenberg

Summary. Three to four ovaries from rats on the day of di-oestrus I were placed in perifusion culture at 10:30 h and exposed to (1) no gonadotrophin (in-vitro controls); (2) tonic FSH (200 ng RP-1/ml); (3) tonic LH (30 ng RP-1/ml); (4) tonic FSH + tonic LH; or (5) tonic FSH plus hourly pulses of LH (amplitude = 50 ng/ml). The total amount of LH administered was 3060 ng RP-1 regardless of mode of delivery. After culture for 3 h, the ovaries were prepared for histological analysis. Compared to in-vitro controls, tonic LH stimulation increased the number of follicles with > 1·6 × 105 granulosa cells (P < 0·05); it was estimated that each follicle in the larger size class increased by 5·5 ± 2·7 × 104 cells. Tonic FSH or tonic FSH + tonic LH treatment did not promote growth into the 1·6 × 105 cell class. In the presence of tonic FSH, hourly LH pulses induced follicular growth similar to that observed after tonic LH treatment. The data demonstrate that LH promotes the growth of follicles in vitro. FSH modulates this stimulatory action of LH, allowing it to be expressed when LH is administered in hourly pulses.

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R Gosden and M Nagano

Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.

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S. K. Roy and G. S. Greenwald

Summary. Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1–5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 μg progesterone or oestradiol-17β in serum-free defined medium and exposed to 1 μCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1–4 (1–4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5–6 layers granulosa cells and developing theca) and 6 (7–8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1–5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7–8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1–4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.

Keywords: preantral follicle; hamster; long-term culture; DNA synthesis; gonadotrophins

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R A Anderson

Human fertility is dependent on maturation of germ cells through meiosis and their association with supporting cells, which in the female are also the source of sex steroids. These processes are sensitive to both chemotherapy and radiotherapy thus can be damaged by anti-cancer treatments. The uterus is also sensitive to radiotherapy. Our understanding of and the ability to manipulate fertility has increased together with survival rates from many cancers, particularly those affecting children, younger men, and women. The growth of interest in fertility preservation for cancer patients is a natural union of these two fields. Sperm banking has been available for many years, and is a recognized and evidence-based option for men that should be available to all. Options for women and pre-pubertal boys and girls are, however, more experimental, other than for women of committing oocytes to fertilization and cryopreservation as embryos. This Focus Issue of Reproduction aims to address the current status of some of the clinical and laboratory aspects of this burgeoning subspecialty to highlight not only areas of progress but also areas of uncertainty where future developments are required to allow the provision of accurate information, and safe and effective treatments.

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M. Mattioli, G. Galeati, B. Barboni and E. Seren

Intracellular concentrations of cyclic AMP (cAMP) were measured in pig oocytes maturing in vivo or in vitro. Maturation in vivo was induced with 500 iu hCG administered to gilts treated with pregnant mares' serum gonadotrophin (PMSG). Although PMSG did not affect cAMP concentrations (basal values, 1.69 ± 0.28 fmol per oocyte), hCG induced a transient rise (8.86 ± 1.15 fmol per oocyte 12 h after hCG injection). Similarly, the cAMP concentration rose in oocytes maturing in vitro if the oocytes (surrounded or not by cumulus cells) were co-cultured with the follicle wall in the presence of LH. The same increase in cAMP was obtained when denuded oocytes were co-cultured with mural granulosa cells. Theca cells exhibited only a moderate activity, while cumulus cells were totally ineffective. Granulosa cells exposed to LH lost their stimulating influence after 24 h of culture. In the presence of FSH, cAMP production by the oocyte was unaffected by any type of follicle cell. The role of cAMP in the control of oocyte maturation was investigated using dibutyryl cAMP. The presence of dibutyryl cAMP prevented the resumption of meiosis in a dose-dependent manner, but when it was present during the first 12 h of culture only, meiotic progression was accelerated (0 versus 47% of oocytes had germinal vesicles in groups treated with dibutyryl cAMP and control groups, respectively, after 24 h of culture). The results demonstrate that: (i) cAMP concentrations increase transiently in oocytes before the resumption of meiosis; (ii) increased concentrations of cAMP depend on the stimulation of oocyte adenylyl cyclase, possibly by a soluble factor produced by follicle cells exposed to LH; (iii) the increase in cAMP is probably confined to the first 10–20 h of maturation owing to the progressive reduction of the stimulating influence of LH-treated somatic cells; and (iv) a high concentration of cAMP throughout maturation maintains meiotic arrest and a transient increase may facilitate meiosis.

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M. A. Driancourt, P. Guet, K. Reynaud, A. Chadli and M. G. Catelli

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 ± 0.09%) than in the culture medium (1.3 ± 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp to bovine follicles in vitro depressed aroaromatase altering the ltalue (and Kmossibly the poss value) oVmaxe enzyme. It is proposed that rop 90 is a functional regulator of follicular maturation through its action on aromatase.

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R. H. F. HUNTER, R. A. S. LAWSON and L. E. A. ROWSON

Cytological examination of the oocytes of a number of mammalian species has indicated that they will resume meiosis upon liberation from the Graafian follicle into a simple culture medium (Chang, 1955; Edwards, 1962, 1965a, b). Evaluation of the physiological status of such oocytes after periods of maturation would best be tested by noting their ability to undergo fertilization, cleavage and, ultimately, to produce viable young. Recent experiments have attempted in-vitro fertilization of mammalian eggs matured in culture and, whilst this has apparently yielded some success with human material (Edwards, Bavister & Steptoe, 1969) and in the rabbit (Thibault & Gérard, 1970), the contrary has proved the case in the large domestic species. In many experiments involving cow and pig eggs, sperm penetration of the zona pellucida of artificially matured oocytes was never

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K. G. Gould and C. E. Graham

Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia 30322, U.S.A.

As part of our efforts to increase the amount of material available for study of the fertilization of primate ova in vitro we are trying to utilize prepubertal females treated with gonadotrophins as oocyte donors. In the course of this work it was noted that oocytes recovered from follicles measuring 3-4 mm in diameter behaved in an unexpected manner.

Fifteen prepubertal rhesus monkeys weighing 3-4·5 kg were used. The precise ages of the females were not known, but menstruation had not been seen in any of the animals.

The females were given a daily i.m. injection of 500 i.u. PMSG (Gestyl: Organon) for 5 days before death. The ovaries were removed and oocytes recovered by puncture of the follicles under Ham's F10 medium.

The morphological normality and maturity of the oocytes was checked at ×160 magnification before culture. Granulosa

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K. M. Henderson and P. Franchimont

Summary. The regulation of ovarian inhibin production was investigated using a rat pituitary cell culture system as a bioassay for inhibin activity. Bovine follicular granulosa cells produced inhibin in vitro provided that the culture medium contained serum. The stimulatory factor(s) present in serum is unlikely to be gonadotrophins, because bovine LH and/or FSH failed to stimulate inhibin production when added to medium devoid of serum. Luteinization of granulosa cells in culture was accompanied by a reduction in their inhibin production and an inverse relationship existed between inhibin and progesterone production by granulosa cells. Bovine corpus luteum cells in culture failed to produce detectable amounts of inhibin.

Androgens stimulated granulosa cell inhibin production with testosterone and 5α-dihydrotestosterone being more potent than androstenedione. The androgens did not stimulate inhibin production by luteal cells. Progesterone inhibited granulosa cell inhibin production but oestrogens had no effect. Measurement of steroids and inhibin in fluid from individual follicles indicated that as follicle size increased, concentrations of oestradiol-17β increased, testosterone and inhibin decreased and progesterone remained unchanged. The stimulatory effect of testosterone on inhibin production in vitro together with the parallel changes in follicular fluid concentrations of testosterone and inhibin suggest that ovarian inhibin production in vivo may be controlled, at least in part, through androgens modifying granulosa cell inhibin production. The inhibitory effect of progesterone on granulosa cell inhibin production may be more important in regulating ovarian inhibin production at the time of granulosa cell luteinization and CL formation. The stimulatory effect of androgens on granulosa cell inhibin production might also be a means by which androgens promote follicular atresia.