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J. M. Bedford, T. Mori and S. Oda

In the musk shrew, Suncus murinus, the behaviour of the cumulus—egg complex and its interaction with spermatozoa were unusual in several respects. The cumulus oophorus was ovulated about 15.5 h after mating or treatment with hCG as a hyaluronidase-insensitive matrix-free ball of cells which remained for relatively long periods of about 14 h around fertilized, and for about 24 h around unfertilized eggs. As a probable function of the small number of up to about 10 or 20 spermatozoa that' generally reached the oviduct ampulla from isthmic crypts, there was often a delay of up to 10 h after ovulation before most eggs were penetrated. Soon after ovulation, however, the corona radiata retreated progressively from the zona pellucida, creating a closed perizonal space within the cumulus oophorus. Usually, most spermatozoa that did reach the ampulla were found within a cumulus and generally within that perizonal space. However, whereas the acrosome was intact among the few free ampullary spermatozoa, and in those adhering to the zona of cumulus-free eggs after delayed mating, all spermatozoa seen moving within the cumulus or adhering to the zona of unfertilized eggs had shed the giant acrosome. In accord with current observations in other shrews, the cumulus in Suncus may therefore function not only to sequester spermatozoa, but also as an essential mediator of fertilization – probably by inducing the acrosome reaction. In the absence of the acrosomal carapace that expresses the zona receptors in most mammals, fertilizing Suncus spermatozoa could use an unusual array of barbs on the exposed perforatorium to attach to the zona pellucida.

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J. M. Bedford, T. Mori and S. Oda

The musk shrew Suncus murinus was studied with regard to induction of ovulation, the genesis and role of the vaginal copulation plug, and the behaviour of gametes and embryos within the Fallopian tube. Ovulation occurred about 15 h after ejaculation, which required a mean of 5.2 (range 2–10 intromittent thrusts. Since ovulation occurred also after five thrusts without ejaculation, and after ejaculation without plug formation or sperm deposition, the primary stimulus for this seemed to be the thrust of the penis, the glans of which was covered by a dense field of spines. Neither vasectomized nor prostatectomized males formed a plug at ejaculation, and in the latter case the mean number of spermatozoa reaching the isthmus of the Fallopian tube, the number at the ampullary fertilization site and the rate of fertilisation were lower than in females mated to normal males. Thus both the vesicular gland on the vas deferens and the prostate are essential for formation of the copulation plug, which appears to enhance sperm transport within the female tract. At ejaculation, ≤ 106 spermatozoa were incarcerated by the plug in the anterior vagina for 6–7 h, by which time a maximal population of several hundred had become established in posterior crypts of the isthmus of the Fallopian tube as small groups of free languidly moving spermatozoa. It remains to be established whether oviductal crypts in this and other shrews have a storage function for spermatozoa or sequester spermatozoa and so regulate the number that reach the fertilization site. Very few spermatozoa reached the ampulla of Suncus. Generally, only one or two spermatozoa had reached the ampulla by 4–5 h, and often less than ten had done so by 5–13 h after ovulation. As a probable correlate, few eggs were penetrated during the first 5 h, with a frequent delay of 10–13 h before most eggs were fertilized. Thereafter, unfertilized eggs were transported through the oviduct at the same rate as developing embryos, which entered the uterus about 85 h after ovulation at the 32-cell stage. There were highly significant differences between the larger KAT/SK strains and smaller OK strain with regard to Fallopian tube length (mean 6.9 mm versus 9.7 mm), as well as the rates of hCG-induced ovulation (5.6 versus 3.25) and of unilateral ovulation (6% versus 50%).

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T. S. Choi, M. Mori, K. Kohmoto and Y. Shoda

Summary. Mouse oocytes matured in vitro in chemically defined medium were not penetrated by spermatozoa. The time required for dissolution of the zona pellucida of such oocytes by α-chymotrypsin was much longer than that for ovulated oocytes. Addition of fetal calf serum to the medium for maturation of oocytes improved the incidence of sperm penetration and shortened the time of enzymic dissolution of the zona pellucida. These results suggest that the low rate of fertilization of oocytes matured in vitro is mainly due to qualitative changes of the zona pellucida, which could be overcome by a factor or factors in fetal calf serum.

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M Nishihara, Y Takeuchi, T Tanaka and Y Mori

The hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator governs intermittent discharges of GnRH into the pituitary portal circulation and, consequently, modulates the pulsatile pattern of gonadotrophin secretion. Electrophysiological correlates of pulsatile gonadotrophin secretion have been demonstrated in the mediobasal hypothalamus of monkeys, rats and goats by recording multiple unit activity. A temporal coincidence between characteristic increases in multiple unit activity and gonadotrophin pulses in the circulation is seen under a variety of physiological and experimental conditions in all three species examined, providing evidence that hypothalamic multiple unit activity originates in the GnRH pulse generator. During a preovulatory gonadotrophin surge induced by oestrogen in ovariectomized animals or occurring spontaneously in intact animals, GnRH pulse generator activity is decelerated, suggesting that it is not involved in generating the gonadotrophin surge. The gonadotrophin surge may be generated by an oestrogen-responsive neuronal complex intrinsically different from the GnRH pulse generator, the electrical operation of which remains unknown.

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M. Koshida, A. Takenaka, H. Okumura and T. Mori

Summary. Immature Wistar rats were induced to ovulate by treatment with PMSG and hCG. Control animals ovulated 43·5 ± 0·36 ova/rat. Intraperitoneal injection of rotenone doses of 0·125, 0·25 and 0·50 mg/kg reduced the ovulation rate to 24·0 ± 3·08, 8·0 ± 0·88 and 1·5 ± 0·44 ova/rat, respectively. The rotenone significantly reduced ovarian cytochrome oxidase activity and progesterone production, but not production of oestradiol or testosterone. Thyroxine treatment at a dose of 5 mg/kg s.c. reversed the rotenone inhibition of ovulation. The results suggest that an increase in mitochondrial respiration is an essential feature of the ovulation process in mammals.

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Y. Mori, M. Tanaka, K. Maeda, K. Hoshino and Y. Kano

Summary. Ovariectomized Shiba goats carrying an oestradiol implant (4–10 pg/ml) were kept under a short-day light regimen (10L:14D; Group 1, N = 4) or a long-day regimen (16L:8D; Group 2, N = 4). Plasma LH concentrations were lower (P < 0·05) in Group 2 than in Group 1 between Days 40 and 200, suggesting an enhanced negative feedback effect of oestradiol on LH secretion under a long-day regimen.

On Days 30, 60, 100, 149 and 279, an LH surge was induced by i.v. infusion of oestradiol for 48 h; the infusion rate was gradually increased from 0·5 (0 h) to 4·1 (48 h) μg/h, thereby mimicking the preovulatory increase of oestradiol secretion. The duration and magnitude of the induced LH surge were indistinguishable between the groups. The latency from the onset of oestradiol infusion to the LH surge was relatively constant in Group 1, 41·1 ± 0·9 h (mean ± s.e.m., n = 17) but was shorter in Group 2 (19·7 ± 3·7h, P < 0·05) on Day 149; less oestradiol was therefore required for induction of the LH surge (27·4 vs 89·7 μg, P < 0·01), suggesting an increased sensitivity to the oestradiol positive feedback under a long-day regimen.

These results might be interpreted to indicate that the hypothalamic–pituitary axis of the goat becomes hypersensitive to the positive as well as the negative feedback effect of oestradiol under long-day conditions.

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N. Kataoka, S. Taii, M. Kita and T. Mori

A novel method for purifying dispersed porcine theca cells, with less than 3% granulosa cell contamination, was developed by the repeated use of mechanical and enzymatic procedures. The steroidogenic criteria used for the identification and purity evaluation of both theca and granulosa cells were also improved. Purified theca and granulosa cells from medium-sized follicles displayed steroidogenic differences when they were cultured in the presence of 10% fetal bovine serum: (1) the theca cells synthesized oestradiol (239.1 ± 35.1 pg ml−1 per 2.5 × 105 cells in 40 h), but the granulosa cells did not synthesize it unless aromatizable androgens were added; (2) theca cells synthesized androstenedione (73.2 ± 14.4 ng ml−1 per 2.5 × 105 cells in 40 h), but granulosa cells did not; (3) FSH did not affect progesterone production in theca cells; (4) the theca cells secreted androstenedione for up to 48 h; and (5) FSH significantly stimulated progesterone production in granulosa cells during a culture for 40 h (P < 0.05), but not during culture for 12 h. The lack of response to FSH was used as a reliable, functional indicator of the purity of porcine theca cells. However, this criterion proved not to be useful for cells cultured for 12 h; porcine FSH had no effect on the progesterone production of theca cells co-cultured for this time with as many as 20% granulosa cells. However, after co-culturing for 40 h, this criterion resulted in the detection of only 3% granulosa cell contamination. Lack of response to FSH is a sensitive and reliable criterion for evaluating the purity of porcine theca cells, as long as FSH responsiveness of granulosa cells is fully confirmed.

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M Fahrudin, T Otoi, NW Karja, M Mori, M Murakami and T Suzuki

The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.

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P. A. Racey, T. A. Uchida, T. Mori, M. I. Avery and M. B. Fenton

Summary. Copulation lasted for up to 46 min in little brown bats. Spermatozoa were stored in both the uterus and the utero-tubal junction, although intimate relationships between spermatozoa and the epithelium were particularly evident in the utero-tubal junction, and were established at the beginning of the period of sperm storage. Polymorphonuclear leucocytes were present in all uteri irrespective of whether or not they had been inseminated but were not generally present in the utero-tubal junction or oviduct. Engulfment of spermatozoa by the epithelial cells of the utero-tubal junction and by polymorphonuclear leucocytes in the uterine glands was evident soon after copulation. It is suggested that this may effect the removal of defective spermatozoa and allow luminal spermatozoa access to the spatially restricted storage sites.

Uninseminated female bats attempted to elicit copulation from torpid males, and were also observed adjacent to copulating pairs. Female bats also uttered copulation calls.

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M. Kita, S. Taii, N. Kataoka, A. Shimatsu, K. Nakao and T. Mori

The activity of GnSI/AF was measured in pig follicular fluid (pFF) from 58 individual follicles of various sizes, by bioassay using rat pituitary cells, to investigate the relationship between gonadotrophin surge inhibiting/attenuating factor (GnSI/AF) activity and follicular development. In addition, the correlation between GnSI/AF and inhibin activities and the content of sex steroids (oestradiol, progesterone and testosterone) of follicles was examined. The activity of GnSI/AF in pFF varied significantly (0.155–1.69 U μl−1) with size of the follicle. The activities (mean ± sem) were intermediate and constant in follicles with diameters from 3 to 5 mm (0.583 ± 0.080 U μl−1, n = 24), were higher and reached the highest value in follicles with diameters between 6 and 8 mm (0.863 ± 0.068 U μl−1, n = 21), and were lower, reaching the lowest value in follicles with diameters of 9 and 10 mm (0.401 ± 0.089 U μl−1, n = 13). In contrast, inhibin activity was almost constant during the development of follicles, although individual values varied from 0.9 to 2.5 U μl−1. For follicles with diameters of 4–8 mm, inhibin activity was 1.754 ± 0.042 U μl−1 (n = 39); activity was higher in the smallest follicles with diameters of 3 mm (2.063 ± 0.015 U μl−1, n = 6) and was lower in follicles with diameter of 9 mm, reaching the lowest value in follicles with diameter of 10 mm (1.176 ± 0.068 U μl−1, n = 7); inhibin activity was not significantly correlated with GnSI/AF activity. Steroid concentration increased in a similar pattern to that of GnSI/AF activity, but no marked decrease was noticed in the large follicles. These characteristic changes of the activity of GnSI/AF of follicles during development, especially in the preovulatory stage, suggest that GnSI/AF plays a role in the ovulatory process as an ovarian regulator for the timely occurrence of the LH surge.