In chicken embryos, there is a difference between the sexes in the onset of lutropin receptor mRNA expression in the gonads. The effects of oestrogen on lutropin receptor expression were studied to investigate the mechanism controlling this difference. Lutropin receptor mRNA expression was detected in the ovaries of sesame oil-treated control female embryos on day 12 of incubation, while no expression was found in the testes of the male controls. Oestradiol administration to genetically male embryos before sexual differentiation resulted in gonadal sex reversal which was characterized histologically by the proliferation of cortical cords and the presence of lacunae. Lutropin receptor expression was detected in the feminizing testis on day 12 of incubation. Administration of aromatase inhibitor (CGS 16949 A) to genetically female embryos before sexual differentiation inhibited the formation of cortical cords, although a relatively weak expression of lutropin receptor was detected. These results indicate that early expression of the lutropin receptor is regulated by oestrogen.
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Y Akazome, T Abe, and T Mori
The gonad is an endocrine organ secreting sex hormones and also a target of pituitary gonadotrophins. The expression of mRNAs encoding LH receptor (LHR), FSH receptor (FSHR), P450c17 and P450aromatase in the developing gonads of embryos between day 4 and day 6 of incubation was determined using a RT-PCR to elucidate the chicken gonad as a target organ of gonadotrophins. Although expression of mRNAs encoding LHR, FSHR and P450c17 was detected at day 4 of incubation in both sexes, mRNA encoding P450aromatase appeared at day 6 in female embryos only, indicating that mRNAs encoding gonadotrophin receptors can be identified before sexual differentiation. Quantitative PCR analysis revealed that expression of mRNA encoding LHR and FSHR remained low in male gonads from day 4 to day 6 of incubation, whereas they increased on day 6 in female gonads. The sexual dimorphism in the expression of mRNAs encoding LHR and FSHR was confirmed in the sexually differentiated gonads of embryos at day 12 of incubation (LHR in ovary ratio LHR in testis = 7 ratio 1; FSHR in ovary ratio FSHR in testis = 9 ratio 1).
T. Mōri and T. A. Uchida
Summary. Only a very few spermatozoa were found in the ampulla of the oviduct just after ovulation. The spermatozoa lost both the acrosome cap and the equatorial segment while passing between the cumulus cells surrounding an ampullar egg; many such spermatozoa were found in the perivitelline space. One spermatozoon was seen in contact with the plasma membrane of the ovum in the metaphase of the second meiotic division. Excess spermatozoa in the perivitelline space were phagocytosed by pseudopodial protrusions from the blastomere surface.
T. Mōri and T. A. Uchida
Summary. In the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, ovulation occurred spontaneously with the disappearance of the granulosa layer and germinal epithelium at the apex of the stigma which was formed simultaneously with expulsion of the first polar body, and with the subsequent bleeding from the capillary lumina of the theca interna. After ovulation the rupture point was plugged by luteinizing granulosa cells and overgrown by newly regenerated cells of the tunica albuginea and germinal epithelium.
T. Mori and T. A. Uchida
Summary. Live spermatozoa were first stored in the colliculus tubaricus of the uterotubal junction (UTJ) on both sides, Spermatozoa then ascended to the caudal isthmus just before ovulation. Spermatozoa with intact acrosomes in the caudal isthmus, which had their heads orientated towards the cephalic isthmus, were seen amongst groups of epithelial cilia in parallel to the epithelium; on the other hand, acrosome-less spermatozoa that were not attached to the epithelium were orientated irregularly in the lumen. A few acrosome-intact spermatozoa were found in the left ampulla at the time of ovulation of the single egg on the left side. Most of the spermatozoa failed to reach or survive in the ampulla of the right oviduct and leucocytic phagocytosis was restricted to this side.
T. Mōri and T. A. Uchida
Summary. In the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, spermatozoa in contact with the microvilli of non-ciliated cells in folds in the uterotubal junction appeared normal, while spermatozoa remaining in the uterus degenerated and were engulfed by a massive invasion of polymorphonuclear leucocytes. No leucocytes were seen in the uterotubal junction area. It is suggested that the spermatozoa in this area are being stored, probably until ovulation occurs.
H Iida, T Kaneko, S Tanaka, and T Mori
The spermatozoa of the musk shrew, Suncus murinus, have a fan-like giant acrosome with a diameter of approximately 20 mm. The aim of this study was to investigate how this giant acrosome is constructed in the musk shrew spermatid and, in particular, how the Golgi apparatus involved in acrosome formation behaves. The behaviour of the Golgi apparatus was monitored by confocal laser scanning microscopy with antibody against a Golgi-associated Rab6 small GTPase. In the early Golgi phase, small Golgi units, the Golgi satellites, localized as a large aggregate in the juxtanuclear cytoplasm. As acrosome formation progressed, the Golgi satellites gradually dispersed, associated with proacrosomal vesicles and an acrosomal vesicle, and finally became distributed as multiple small units over the whole surface of an acrosomal cap in the round spermatid. The mode of acrosome formation in musk shrews was distinctly different from that in rats and mice, in which the Golgi apparatus remains as a single unit throughout acrosome formation. In musk shrews, the proacrosomal vesicles formed successively by the Golgi satellites coalesced, one after another, into a potential acrosomal vesicle. This process may result in further enlargement of the acrosome. The results of the present study indicate that Golgi satellites are necessary for the biogenesis and development of the giant acrosome in musk shrew spermatozoa.
I. Takai, T. Mori, Y. Noda, and T. Nishimura
Summary. Rabbit antiserum raised against isolated human ova produced an intense immunofluorescence and a precipitate on the outer surface of human and porcine zonae pellucidae. The antiserum reacted with none of 22 human tissues nor with any of 4 body fluid components by immunofluorescence or immunodiffusion analysis, but the antiserum agglutinated AB erythrocytes after absorption with O erythrocytes. The anti-zona activity was achieved by higher titres for human than for porcine zonae. Immunofluorescence on porcine zonae was completely abolished by absorption with porcine ova, whereas a weak but definite fluorescence remained on human zonae. These findings indicate that the human zona pellucida consists of at least three distinct components; (1) a specific antigen(s) shared by human and porcine zonae, (2) an antigen(s) specific to human zonae, and (3) a non-specific antigen(s) associated with the blood group substances.
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.
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%).