Showing papers in "Reproduction in 1969"

In vitro capacitation of hamster spermatozoa by follicular fluid

Abstract: When hamster epididymal spermatozoa were incubated in vitro in the presence of follicular fluid in Tyrode's solution for 3 hr or more, they became fully capacitated. The majority of the capacitated spermatozoa had lost the acrosome cap, and they displayed an extremely vigorous motility. When placed in contact with freshly ovulated eggs in vitro, the spermatozoa started to enter the zona pellucida of the eggs

Identification and subcellular localization of the enzymes effecting penetration of the zona pellucida by rabbit spermatozoa.

Abstract: A sucrose density gradient centrifugation procedure for separating the subcellular components of washed and sonicated rabbit epididymal spermatozoa has been developed and applied to the subcellular localization of several sperm enzymes, including the acrosomal enzymes involved in penetrationofthe zona pellucida by the spermatozoon. These zona penetration enzymes were identified as hyaluronidase and a trypsin-like enzyme. They were extracted as a single complex, which sedimented in a sucrose gradient in the vicinity of proteins with molecular weights of 59,000. The combined action of these two enzymes in the acrosomal extract produces a rapid and complete dissolution of the

A histological and histochemical examination of implantation and early placentome formation in sheep.

Abstract: Embryo attachment and implantation in the ewe have been examined with a view to (a) elucidating the interrelationships of the trophoblast and the uterine epithelium, and (b) defining accurately the time relationships of the various phenomena. Embryonic attachment occurred during the 15th day of pregnancy and cytological changes within the uterine caruncular epithelium that appeared on the following day and were completed within a week, resulted in the formation of plaques of multinucleate symplasm and the death of some maternal epithelial cells. High levels of acid phosphatase activity, considered to demonstrate the release of lysosomal hydrolases, were associated with the modification of the maternal epithelium and with the later trophoblastic invasion of the uterine stroma. Alkaline phosphatase activity present in the apical cell membranes of the opposing trophoblast and uterine epithelium is considered to be associated with carbohydrate metabolism and the production of trophoblastic fibrinoid. No evidence was obtained to support proposals that trophoblastic binucleate giant cells have a major role in modifying the uterine epithelium through their phagocytic activity or as a source of the cryptal epithelium. The cryptal epithelium during the first 6 weeks ofpregnancy appears to be derived from the symplasmic plaques of maternal epithelium.

Comparison of vaginal tolerance tests of spermicidal preparations in rabbits and monkeys.

Abstract: In order to decide whether the rabbit method or the rhesus monkey test is better suited for routine vaginal tolerance tests of spermicidal preparations, combined trials employing both techniques were carried out in the two laboratories in which the tests had been developed. A `double-blind' experimental design was used in which three unknown, coded compounds were tested jointly in both laboratories and evaluated independently and reciprocally after transatlantic exchange of the resulting histological material. Both test methods and the scoring systems employed in the assessment of findings are described and illustrated by representative photomicrographs. There was good agreement between both methods for two of the three preparations tested. For the third preparation, the rabbit test results were more consistent with the available clinical data than those of the monkey test. It was concluded that the rabbit technique is more sensitive than the monkey test. Since it has several obvious practical advantages over the latter, it is proposed that the rabbit vagina test should be generally adopted as the standard method for establishing the local tolerance of

Numbers of spermatozoa in the genital tract after artificial insemination of progestagen-treated ewes.

Abstract: An experiment is described in which spermatozoa were recovered from genital tracts of ewes either at the first oestrus following progestagen synchronization or at a normal oestrus. Recoveries 1, 12, 24, 36 and 48 hr after the insemination of 500\m=x\106 spermatozoa show that synchronization with intravaginal sponges impregnated with the synthetic progestagen 17\g=a\-acetoxy-9\g=a\-fluoro-11\g=b\\x=req-\ hydroxypregn-4-ene-3,20-dione (Cronolone, Searle) alters the numbers and distribution of spermatozoa throughout the female genital tract at the first oestrus after treatment. One hour after insemination the total numbers of spermatozoa recovered, and their distribution between the various regions of the tract, differed little between progestagen-treated and untreated ewes. Thereafter the numbers recovered from treated ewes were significantly fewer than from untreated, and their distribution differed. Significant differences appeared in the vagina at 12 hr and in the cervix and uterus at 24 hr and thereafter this difference was maintained. The pattern in the Fallopian tubes followed that of the uterus. At 24 hr the mean numbers of tubal spermatozoa recovered were: synchronized\p=m-\900; non-synchronized\p=m-\8200(P< 0\m=.\001). Both transport and survival of spermatozoa may be deleteriously affected in the reproductive tract of the progestagen-treated ewe.

Fertility following egg transfer in the cow; effect of method, medium and synchronization of oestrus

Abstract: Summary. A total of ninety-two heifers was used in a series of four experiments designed to investigate the effect of method of egg recovery, transfer, storage medium and degree of synchronization between donor and recipient compatible with fertility. Experiments 1 and 2 involved the transfer of thirty-three eggs in homologous serum to eighteen recipients. In half of these, the eggs were transferred surgically and in the other half non-surgically. No pregnancies resulted from these transfers. In Experiments 3 and 4, a total of seventy-six eggs was transferred to forty recipient heifers using TCM 199 as the transfer and storage medium. Twenty of these transfers were made surgically, the eggs also being recovered by surgical means in eleven cases and by slaughter in nine. The pregnancy rate obtained was 91% for surgically recovered and transferred eggs and 33% for eggs recovered at slaughter and then transferred surgically. The remaining twenty animals received eggs, half of which were recovered surgically and half at slaughter, and all then transferred by non-surgical means. It is concluded that homologous serum is an unsuitable medium for egg transfer but that TCM 199 is highly satisfactory. The recovery of eggs from slaughtered animals appears less satisfactory than if they are recovered in vivo. A degree of variation from exact synchronization of \m=+-\2days can be tolerated.

Peripheral plasma progesterone levels during the bovine oestrous cycle.

Abstract: Progesterone was determined daily in the peripheral plasma of six cows for a total of seven complete oestrous cycles. Progesterone levels ranged from less than 0\\m=.\\5ng/ml plasma during the follicular phase to 6\\m=.\\6ng/ml plasma (6\\m=.\\1to 10\\m=.\\2ng) at peak luteal phase. Progesterone levels in cows with 21-day cycles increased rapidly from Day 3 to Day 8 (oestrus = 1) with a much slower rate of increase from Day 8 to Day 17. These cows showed a progesterone decrease of more than 50% from the previous day on Days 18 (two cows), 19 (one cow) and 21 (two cows). Two other cows with cycles of 22 and 23 days' duration both had a similar decline on Day 20. A variable time interval of 1 to 5 days was observed between the decline ofprogesterone and the occurrence ofoestrus. These data indicate that considerable variation may exist among cows as to time requirements for follicle development and maturation. Monitoring ofperipheral levels ofprogesterone is suggested as a means ofstudying corpus luteum function.

A second experiment with heterospermic insemination in cattle.

Abstract: Friesian cows were inseminated with semen mixtures containing equal numbers of spermatozoa from a Friesian and a Hereford bull. The five bulls of each breed gave twenty-five possible combinations. The paternity of calves was established by inspection of colour and conformation. Heterospermic indices were calculated to express the relative ability of sires to father offspring after mixed insemination. There were significant differences between the heterospermic indices of bulls, the maximum observed difference being twenty-one-fold. The indices were consistent over two series. The homospermic index was defined as the 16-week non-return rate after normal single first inseminations. The heterospermic index established differences between bulls more efficiently than the homospermic index; one estimate showed that the heterospermic method needed less than 1/170th the number of inseminations required by the homospermic method. The homospermic index was predictable from the heterospermic index, the regression coefficient having a significance level of 0\m=.\05> P > 0\m=.\025.The initial spermatozoan concentration of a bull's semen (before dilution) was highly correlated with the heterospermic index. Measures of semen quality based on the morphology and staining affinity of spermatozoa predicted heterospermic and homospermic indices non-significantly but in the right direction.

Delayed fertilization and chromosomal anomalies in mouse embryos.

Abstract: Chromosomes were studied in mouse embryos and foetuses recovered after superovulation. The spontaneous incidence of chromosomal anomalies was 2\m=.\6%;a delay of 7 to 13 hr before fertilization produced 3\m=.\9%anomalies. The most striking feature was a nine-fold increase in the incidence of triploidy after delayed fertilization. The chromosomal anomalies included isochromosomes and minute chromosomes. Some anomalous individuals survive implantation and the fate of such foetuses is discussed.

The incidence of anovular cycles in women.

Abstract: Basal body temperature records were used to estimate the frequency of anovular cycles with a shortened corpus luteum phase in 3264 cycles of girls and women of all ages. In each age group the number of anovular cycles cycles with a shortened corpus luteum phase and the number of normal cycles were recorded and converted into percentages. Between the ages of 12-14 years there was a high prevalence of anovular cycles. In later age groups an almost continuous reduction of anovular cycles was found until the minimum was reached in the 26-30 year age group. After the 40th year the proportion of anovular cycles again increased. From the perspective of the gynecologist who is primarily concerned about the problem of infertility in married women the cycles with abnormally shortened hyperthermic phases were almost of greater importance as they were another cause of sterility. In the age group which contained the greatest number of infertility patients the 25-40 age group the cycles with shortened corpus luteum phase were significantly more frequent than the anovular cycles. In more than 1200 infertility patients cycles with shortened hyperthermic phase were 2 1/2 times as numerous as anovular cycles. In rhesus monkeys a predominance of anovular cycles during the summer months was observed. To learn about the distribution of anovular cycles and cycles with shortened corpus luteum phase basal temperature records of 95 women from the 1st to the 4th menstruation postpartum were taken. 63 (22%) of 285 cycles were anovular and in 91 32% cycles the hyperthermic phase lasted less than 10 days. This means that during the 1st few cycles postpartum the temperature records began with onset of the 1st menstruation. The conclusions here were drawn from the 2nd 3rd and 4th cycles. 46% of the cycles were normal and 54% were diagnosed as insufficient. The 1st cycle was biphasic in only a few instances. The most striking conclusion is the high percentage of insufficient cycles in the early years after the 1st menstruation. This explains the comparative infertility of young girls.

Relationship of a trypsin-like enzyme in rabbit spermatozoa to capacitation.

Abstract: The existence of a trypsin-like enzyme (TLE) in acrosomes ofepididymal spermatozoa was confirmed and was further demonstrated to be present in acrosomes of ejaculated and capacitated spermatozoa. TLE rapidly removes the zona pellucida of the ovum. Extracts of acrosomes of ejaculated spermatozoa contain an inhibitor that is separated from the TLE by purification of the TLE. The inhibitor of TLE is also present in seminal plasma. This enzyme\p=m-\ enzyme inhibitor relationship appears analogous to the corona-removing enzyme-decapacitation factor relationship and part of capacitation very likely involves removal of the inhibitor from TLE and decapacitation factor from the corona-removing enzyme. TLE is inhibited by soybean trypsin inhibitor and less effectively by mercaptoethanol.

Implantation and development of mouse eggs transferred to the uteri of non-progestational mice.

Abstract: Four-day-old mouse embryos were transferred to the uterine lumen of virgin cyclic and ovariectomized mice; the eggs 'implanted' and developed only in mice whose endometrium was previously traumatized with a glass scraper. The histology of the mechanically induced implantation sites is described and similarities to normal implantation sites are discussed. Implanted embryos developed only to stages equivalent to 5 to 9 days of normal pregnancy, but the trophoblast continued to proliferate and invaded the endometrium, eroding maternal blood vessels and distending the uterus. In five of eight ovariectomized mice, plaques of decidual-like cells were found near the trophoblast 7 to 12 days after transfer.

Plasma levels of testosterone in male rabbits following copulation.

Abstract: It is well established that the rabbit ovulâtes in response to mating and a marked ovarian release of 20a-hydroxypregnen-4-en-3-one will occur shortly after coitus in this species (Hilliard, Archibald & Sawyer, 1963). This change in progestin secretion following mating corresponds with the increase of gonado¬ trophin in peripheral blood (Hilliard, Hayward & Sawyer, 1964), and the release of interstitial cell stimulating hormone (icsh) and prolactin from the pituitary gland (Desjardins, Kirton & Hafs, 1967). Moreover, Taleisnik, Caligaris & Astrada (1966) noted a decrease in pituitary content of icsh and an increase in plasma icsh in female rats following copulation. The level of ascor¬ bic acid in the ovary of these animals was lower than in control rats. The hormonal response of the male gonad to copulation is less overt and has received little attention. Copulation will increase the blood levels of icsh and decrease the pituitary concentration of the hormone in the male rat (Taleisnik et al., 1966). The secretion of androgenic steroids into the spermatic venous blood of anaesthetized rabbits appears to increase shortly after coitus (Endroczi, 1962) and this increase is not found in male rabbits with hypothalamic lesions. Since testosterone is the major androgen secreted by the male gonad, it was deemed important to study fluctuations of this hormone in the peripheral blood of the rabbit following coitus.

Variations in foetal weight and vascular supply along the uterine horn of the pig

Abstract: Summary. Weights of foetuses in ninety pigs were studied in relation to their positions in the uterine horn. Position had no effect when there were some five or fewer foetuses in the horn but as the number increased, those at the ends tended to have an increasing advantage over those in the middle and the foetus at the ovarian end tended to have an increasing advantage over the foetus at the cervical end. Percentage differences in foetal weight were substantially constant over the period of the pregnancy studied, namely 31 to 113 days after conception. Differences in foetal weight between positions could not be explained in terms of the uterine vascular architecture.

Characterization and isolation of a sperm-coating antigen from rabbit seminal plasma with capacity to block fertilization.

Abstract: The relationship between the sperm-coating antigens of rabbit seminal plasma origin and the characterization of the decapacitation factor was studied using a gar-gel diffusion immune-electrophoresis chromatography on Sephadix G-200 and polyacrylamide vertical gel electrophoresis. Spermatozoa incubated in the uterus for 11 hours yielded an 85% fertilization rate and hence were capacitated. Treatment of the capacitated spermatozoa with rabbit seminal plasma resulted in a 38% fertilization rate. It was concluded that a sperm-coating antigen of seminal plasma origin possessed biological activity for blocking fertilization. It was found using the above techniques that the sperm-coating antigen was a glycoprotein of approximately 170000 molecular weight migrated in an electric field similar to a serum slow beta-globulin and was still present in the seminal fluid of vasectomized males. The sperm-coating antigen was absent from inactive upper supernatant fluid fraction of seminal plasma after 4 hours of ultracentrifugation at 105000 g and was present in the active ultracentrifugal pellet.

Composition of rabbit oviduct fluid in ligated segments of the Fallopian tube.

Abstract: Forty-six, mature, New Zealand white does were used for collection of fluid from the Fallopian tubes. Ligation of the tubes into four isolated segments was carried out with minimal disturbance to the blood supply, using an operating microscope. The volume of fluid collected decreased from the fimbriated end to the utero-tubal junction. Analysis of the chemical constituents of the fluid from the first three segments showed that the concentration of sodium, bicarbonate, inorganic phosphate, proteins and lactic acid increased significantly from the fimbriated end to the utero-tubal junction, while chloride concentration decreased significantly. These results support the possibility that the different segments of the oviduct may play specific roles in early events of reproduction.

Preliminary observations on the reproductive effects of the pineal gland in blinded, anosmic male rats.

Abstract: Removal of the eyes (Reiter, 1968) or of the olfactory bulbs (Orbach & Kling, 1966) slightly delays gonadal maturation in rats. The gonadal inhibitory effect of blinding is prevented if animals are pinealectomized (Reiter, 1968). It was found recently that if weanling male rats have both their eyes and olfactory bulbs removed, the testes are less than half their normal size at 70 days of age; this entire effect is reversed if the animals also have their pineal gland removed. This indicates that another external factor, in addition to the photo-period, may influence directly or indirectly the antigonadotrophic activity of the pineal gland. The experiment was repeated and the following data illustrate some interrelationships between olfaction, photo-period, the pineal gland and reproduction. Sixty-four 21to 23-day-old male Sprague-Dawley rats were divided into

Identification of the midpiece and tail of the spermatozoon during fertilization of human eggs in vitro.

Abstract: Positive identification of the sperm midpiece and tail in the vitellus establishes beyond reasonable doubt that pronucleate eggs are undergoing fertilization. Previously Edwards, Bavister & Steptoe (1969) tentatively identified sperm midpieces in pronucleate human eggs fertilized in vitro. Unequivocal evidence of midpieces and tails in eggs undergoing fertilization is now presented. Oocytes recovered from two ovaries excised 3 hr previously were cultured in

Blood pressure and respiratory patterns during human coitus

Abstract: Changes in both blood pressure and in the pattern of respiration were investigated during human coitus. The studies were carried out on 1 married couple aged 37 (male) and 32 (female) with 10 years mutual coital experience in the habitual environment of their own bedroom. The absence of observers introduced a minimum of psychological artifacts into the situation of natural coitus. During coitus the female assumed the supine position in all experiments and efforts were made to emulate the coital pattern which was most typical for the couple. In experiment 2 systolic blood pressure was measured by a Winston blood pressure follower. During Experiment 2 a Benedict-Roth spirometer with C02 absorption was used to obtain 2 male and 2 female spirographs during 4 separate coitions. The recordings were never made simultaneously for both male and female. A peak of blood pressure and a sudden change in respiratory pattern were noted at the male and female sexual climax. Venous blood taken immdiate before coitus from the female has the normal burgundy hue but immediately following coitus blood drawn from the vein took on the color of arterial blood (a bright scarlet).

Effects on spermatogenesis in guinea-pigs, rabbits and sheep after their immunization with sexual organ fluids of bulls.

Abstract: The effect on spermatogenesis of intratesticular and subcutaneous immunization with genital-tract fluids of bulls was studied in sexually mature guinea pigs rabbits and sheep. Considerable degenerative changes and aspermatogenesis were found in the testis of guinea pigs and rabbits which received an intratesticular injection of the seminal vesicle fluid of bulls (SVFB). The infection of the ampullar fluid of sexually mature bulls (AFB) into guinea pigs also had the same effect. In the majority of animals only the injected testis showed degenerative changes while the other remained unaffected. The changes following intratesticular injection of SVFB were usually irreversible. Subcutaneous (sc) injection of SVFB was followed in some animals by a significant decrease in the index weight of testes (compared with controls) and aspermatogenesis. Damage was not alleviated when SVFB was absorbed by ampullar spermatozoa. In rams the damage to the testes was of a temporary reversible character. In the guinea pigs and rabbits it appeared that the damage caused by sc injections of SVFB was usually temporary. In all animals immunized subcutaneously identical circulating antibodies were found as were in animals with damaged testes and in animals with normal testes. They did not cause spermagglutination immobilization or spermatotoxic reactions. No circulating antibodies could be found in guinea pigs and rabbits immunized by a single intratesticular injection.