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  • Author: G. S. GREENWALD x
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J. T. McCORMACK and G. S. GREENWALD

Summary.

Measured by radioimmunoassay, the peripheral plasma oestradiol concentration reached a peak (21·2±1 pg/ml) between 10.00 to 12.00 hours on Day 4 of pregnancy in mice. These values were significantly different from all others measured between 07.00 and 18.00 hours. To determine the physiological significance of this peak, ovariectomies were performed at selected intervals. Ovariectomy at 08.00 hours or earlier usually prevented implantation. The period from 10.00 to 12.00 hours on Day 4 was found to represent a critical time period before which insufficient oestrogen is secreted, in most instances, to initiate implantation.

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H. M. WEITLAUF and G. S. GREENWALD

Summary.

Mature mice were ovariectomized on the 4th day post coitum (Day 1 = vaginal plug) and given no further treatment. Unimplanted (delayed) blastocysts were recovered from these females between 5 and 40 days post coitum. They were tested for viability by determining the proportion that developed into normal foetuses following transfer to Day-4 pseudopregnant recipients.

It was found that the proportion of transferred blastocysts developing into foetuses was similar after delays of from 0 to 10 days. After delays of more than 10 days, the proportion of blastocysts that were capable of developing into normal foetuses decreased; 30 to 32% developed following transfer on Days 5 to 15, 18% with transfer on Day 20, and 3% with transfer on Day 30. The finding that blastocysts can be delayed in the absence of ovarian hormones and yet remain capable of developing into normal foetuses demonstrates that progesterone is not essential for blastocyst survival in mice.

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G. S. Greenwald and R. M. Moor

Summary. An enzymic method for recovering primordial follicles from the pig ovary consists of incubating cortical slices for 2 h with 0·025% collagenase 1A. An average of 185 000 or 419 000 primordial follicles per ovary were recovered from ovaries collected in Cambridge and Kansas, respectively. Following a discontinuous Percoll gradient, primordial follicles can be separated from contaminating somatic cells by mouth pipette or a micromanipulator to collect 100–1500 follicles but for large scale recovery of approximately 30 000 follicles flow cytometry is recommended. Two types of primordial follicles can be distinguished by electron microscopy: peripheral clusters of small oocytes with an incomplete investment of pregranulosa cells and a deeper region of individual oocytes surrounded by a complete layer of pregranulosa cells. The viability of the purified primordial follicles is attested by their ability to synthesize proteins for at least 12 h after incubation with [35S]methionine. Moreover, the primordial follicles showed several polypeptide bands in common with mature oocytes especially with M r of 60 000–90 000 but with considerable differences from somatic cells.

Keywords: primordial follicles; structure; protein synthesis; enzymic dissociation; pig

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S. K. Roy, S. C. Wang and G. S. Greenwald

Summary. As measured by radioreceptor assays, binding sites for FSH and prolactin were present at 09:00 h on the day of pro-oestrus in Stage 1–10 follicles (primary to antral) with prolactin receptors 3–6 times higher than FSH sites in Stages 1–3 (3 layers of granulosa cells). Specific binding sites for hCG were present in Stage 1 and 2 follicles (2 layers of granulosa cells) but thereafter their distribution was erratic and they were not consistently detectable until Stage 5, when thecal cells first appeared. Using topical autoradiography, specific binding for FSH was evident in Stage 1–4 follicles (4 layers granulosa cells) whereas specific hCG-binding was not. After the preovulatory gonadotrophin surges, by 21:00 h on pro-oestrus, FSH receptors declined in Stages 5–10, prolactin receptors fell in Stages 8 and 10 (small and large antral follicles) and hCG receptors were reduced in Stages 7 (start of antral cavity) to 10. On the morning of oestrus, for follicles from Stage 4 onwards, receptor numbers usually returned to levels found at 09:00 h on pro-oestrus. At oestrus, the few remaining Stage 10 follicles were all atretic and contained significantly reduced FSH and prolactin receptors but numbers of hCG binding sites comparable to those at 09:00 h of pro-oestrus. These results provide evidence of gonadotrophin receptors in small primary and secondary follicles which is consistent with increased DNA synthesis in small hamster follicles on the afternoon of pro-oestrus and on the morning and afternoon of oestrus. Periovulatory changes in gonadotrophin concentrations may therefore affect early stages of folliculogenesis.

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X. Wang, A. R. Kole and G. S. Greenwald

This study was designed to determine whether the major site of eCG neutralization by an antiserum to the hormone is at the peripheral or ovarian level. Hamsters hypophysectomized at oestrus were injected s.c. with 25 iu eCG. Three days later, preovulatory follicles were dissected and cultured for 5 h and the medium was changed every hour. At the end of the first hour of incubation, oestradiol and androstenedione accumulation was high, with a sharp drop over the next 4 h, whereas progesterone concentrations did not change over the entire period. Addition of eCG antiserum to the incubated follicles did not affect steroidogenesis. Addition of 1.0 iu eCG in the second hour or every hour sustained oestradiol production at supraphysiological amounts. However, addition of eCG plus eCG antiserum every hour eliminated the stimulatory effects of eCG on oestradiol production. In another experiment, hamsters injected with eCG were treated 3 days later by i.p. injection of eCG antiserum and groups of animals were killed over the next 8 h. Serum samples before and after injecting eCG antiserum were incubated overnight with a goat anti-rabbit immunoglobulin to separate free, unbound eCG from bound eCG. At time zero (before injecting the antiserum) free eCG was increased, but within 1 h after eCG antiserum there was an eightfold decrease of the hormone, and these concentrations were maintained over the next 7 h. The fall in unbound eCG in vivo coincided with the decay in serum oestradiol and androstenedione. These results obtained in vitro and in vivo demonstrate that the long biological half-life of eCG is essential to replenish the supply provided to the ovary constantly to maintain steroidogenesis; eCG antiserum inactivates eCG in serum but not by complexing with eCG at the receptor of the follicular cell surface.