Light and electron microscopic observations on the cervical epithelium of the rabbit: II.

The endocervical epithelium of long-term ovariectomized rabbits treated for 1-10 days with 5 micrograms of estradiol benzoate every 12 hr has been studied by light and electron microscopy. In addition, morphometric data on ciliated and nonciliated cells of rabbits treated for 2, 6, and 10 days are compared to those on untreated ovariectomized, estrous, and ovulatory rabbits. The percentage of ciliated cells increases after ovariectomy to 76.3% and that of secretory cells decreases to 23.7% as compared to estrous controls. Treatment of ovariectomized rabbits with estradiol results in a gradual increase in ciliated and secretory cell area, height, and nuclear area. After 10 days of treatment, cell areas are significantly larger than those in the ovulatory or estrous controls; cell height and nuclear areas have returned to preovariectomized levels; and the percentages of ciliated and secretory cells have reached those of estrous levels. Estradiol stimulates mitotic division of secretory cells but affects ciliogenesis minimally. In ciliated cells, estradiol treatment results in a modest increase in polysomes and granular endoplasmic reticulum and in striking increases in the size of the Golgi complex and in the number of lipofuscin bodies as compared to those in the ovariectomized controls. In secretory cells, estradiol treatment brings about an increase in the numbers of polysomes, Golgi complexes, and cisternae of the granular endoplasmic reticulum, in the sizes of the nucleoli, and in the amount of euchromatin. Secretory granules appear in some cells after 2 days of estradiol stimulation and increase in number through 10 days of treatment. Perinuclear granules are more pleomorphic and heterogeneous in structure and more numerous in the 6- to 10-day-estradiol-treated than in ovulatory animals, and they may function as lysosomes degrading excess secretory product. Deep apical concavities of the secretory cells occur most often after 2 and 6 days of treatment.

Light and electron microscopic observation on the cervical epithelium of the rabbit. I.

The light and electron microscopy of the cervical epithelium of ovulatory, estrous, and long-term ovariectomized rabbits have been studied to determine what structural changes occur under different hormonal conditions. The percentage of nonciliated secretory cells is 49.6 in ovulatory, 43.6 in estrous, and 23.7 in long-term ovariectomized rabbits, and of ciliated cells is 50.2 in ovulatory, 56.2 in estrous, and 76.3 in long-term ovariectomized animals. The values for the ovulatory and estrous rabbits are significantly different at the P less than 0.05 level from those of the ovariectomized animals. In all 3 groups the general ultrastructure of the normal ciliated cells is similar. Interestingly, the Golgi complex is very prominent in all. Glycogen bodies occur frequently only in ciliated cells of ovariectomized and occasionally of estrous animals. Abnormalities in ciliation are quite common in the ovariectomized rabbits. The structure of the nonciliated secretory cells varies appreciably within and between the 3 groups. In these cells from well-developed epithelia of certain ovulatory and estrous animals, the apical cytoplasm contains secretory granules of at least three types. In addition, very irregularly shaped, dense, perinuclear granules occur, which may be another type of secretory granule or lysosomes. As compared to ciliated cells, the secretory cells have less prominent Golgi complexes, more abundant bundles of intermediate filaments, a more extensive glycocalyx on their apical surface, and more heterochromatic nuclei. In comparison to the cells of well-developed epithelia, the nonciliated cells of some other ovulatory and estrous rabbits are less well differentiated with fewer or no secretory granules and less well developed organelles. In the nonciliated cells of the long-term ovariectomized rabbits, there are no secretory or dense perinuclear granules. There is a decrease in the number of organelles that are involved in secretion, in the size of the cells, and in the amount of nuclear euchromatin.

Observations on the solitary cilium of rabbit oviductal epithelium: its motility and ultrastructure.

Solitary cilia have been observed on rabbit oviductal epithelial cells. In tissue cultures of fimbrial epithelium of 3- and 4-day-old animals observed by phase microscopy, most of these single cilia exhibited a vortical or funnel-type movement while others had the usual to-and-fro motility. Primary cilia are usually considered immotile. Transmission electron microscopy of specifically identified single cilia revealed differences between the ciliary shafts and basal bodies of the single cilia as compared to those of mature oviductal ciliated cells. The basal body of the solitary cilium often had at least two triangular, striated, basal foot processes, lacked electron-dense satellite material around its basal end, and occasionally had striated rootlets. In contrast, the cilia of mature ciliated cells had only one basal foot, exhibited much electron-dense satellite material, and lacked rootlets. Cross sections of the single cilia showed patterns of microtubules different from the usual 9 + 2 axonemal complexes of normal cilia and included 9 + 0, 10 + 2 singlets, 7 + 2 doublets, and 8 + 1 doublet and 2 singlets; one did have the usual 9 + 2 arrangement. We postulate that the presence of more than one basal foot process may be responsible for the vortical motility observed. The primary cilia are shorter than normal cilia; the longest one measured was 1.86 micron in length, 0.28 micron in width at its base, and 0.14 micron at its tip. Based on the light-microscopic, scanning-electron-microscopic and transmission-electron-microscopic observations, such solitary cilia were observed more frequently in the oviductal tissues of the 3- to 4-day postnatal rabbits grown in tissue culture and in ovariectomized and ovariectomized/progesterone-treated adult animals than in estrous, ovulatory, or ovariectomized/estradiol-treated rabbits.

In vitro fertilization in the rat: observations on living eggs.

The purpose of this study was to observe and record some of the key events during in vitro fertilization in the rat. Freshly ovulated eggs were incubated with epididymal spermatozoa at 37 degrees C and removed 4.5 to 7.5 hours later for microscopic examination. The head of the fertilizing spermatozoon penetrated the zona pellucida with its long axis perpendicular to the zona: this orientation was maintained during subsequent incorporation into the vitellus. Sperm motility was drastically reduced soon after sperm-egg fusion. Simultaneously, the flagellum, most of which was still outside the zona, assumed a characteristic curved posture. Time-lapse cinematography demonstrated that the vitellus frequently underwent surface movements during the tail incorporation process, suggesting that its cortex was undergoing significant changes. This study presents the first long-term observations on the fertilization of living rat eggs in vitro.

Laser light-scattering study of the effect of washing on sperm motility.

The effect of washing on human sperm motility was measured by means of dynamic laser light-scattering spectroscopy. Semen samples from 24 fertile donors were diluted with Biggers, Whitten and Whittingham (BWW) medium and subsequently centrifuged at one of the following forces: 235 x g, 325 x g, 400 x g, 470 x g, 500 x g, 600 x g, and 800 x g. The duration of centrifugation was 8 minutes for the first wash, 6 minutes for the second wash, and 3 minutes for the third wash. Sperm motility was evaluated in terms of the root mean square swimming speed of the spermatozoa and the mean migration rate of washed spermatozoa in estrous bovine cervical mucus (BCM). It was found that sperm motility and viability were improved when semen samples were washed at 235 x g, even after three washes. However, washing at forces of 600 x g or more reduced sperm motility and also their ability to penetrate cervical mucus in vitro. Repeated washing at forces between 300 x g and 500 x g had little deleterious effect on sperm motility.

Sperm penetration into cervical mucus in vitro. III. Effect of freezing on estrous bovine cervical mucus.

The influence of the storages period on estrous bovine cervical mucus after it was stored in the freezing compartment of the laboratory refrigerator was evaluated by an in vitro sperm penetration test with human spermatozoa, laser light-scattering, and a spinnbarkeit test. Data obtained from the sperm penetration test were analyzed by a mathematical model that correlates the sperm motility with the sperm transport rate and the penetrability of the mucus. The tests showed that estrous bovine cervical mucus can be stored for up to 4 weeks at -12 degrees C without a change in its physical properties. The results of this study strengthen the suggestion that bovine mucus could be employed as a substitute for human cervical mucus.

Optoelectronic transducer for in vivo recording of oviductal contractile activity.

An optoelectronic instrument to record oviductal muscular activity in chronically instrumented animals was evaluated in in vitro and in vivo experiments. The intensity of red light transmitted through the oviduct was modulated by contractions of the oviductal wall producing an optical analog of the mechanical events. Accuracy of the analog was tested by Fourier analysis of signals from mechanical and optoelectronic transducers placed at the same site on the oviduct; the results validated the use of the optical device as a contraction event sensor. Contractions of the tubal mesenteries had less effect on the optical signal than on signals from extraluminal mechanical transducers. Optical and photographic recordings of luminal transport in exposed oviducts showed a correspondence of intraluminal movements to events in the optical contraction signal. This instrument does not alter tubal function, and thus it is an especially useful experimental tool to investigate the role of oviductal muscular activity in fertility.

Cyclic variations in the oviuductal ciliated cells during the menstrual cycle and after estrogen treatment in the pig-tailed monkey, Macaca nemestrina.

The epithelium of the oviduct of the pig-tailed monkey, Macaca nemestrina was studied 1) to determine whether quantitative changes in the number of ciliated, deciliated, reciliating and nonciliated cells occur during the menstrual cycle and under certain experimental conditions and 2) to describe the ultrastructure of the ciliated and ciliogenic cells. The mean percentage of ciliated cells decreased from 48.2 in the fimbriae and 48.3 in the ampullae in the postovulatory stage to 7.7 and 18.8, respectively in the late luteal phase; these changes are significant as determined by Duncan's multiple range test. In the early follicular phase 3.9% of the cells in the fimbriae and 11.2% in the ampullae are ciliated, and the number of ciliogenic (deciliated and reciliating) cells is the highest of any time in the cycle in both the fimbrial (6.3%) and ampullar (8.4%) epithelium. In contrast, although the percentage of ciliated cells in the isthmus varies from 44.4 in the preovulatory phase to 34.3 in the early follicular phase, the differences between the various times in the cycle are not significant. However, in the late luteal phase, the values for the fimbriae and ampullae are significantly different from that of the isthmi. Ciliated cells constitute less than 1% of both the fimbrial and ampullar epithelium 2 3/4 years after ovariectomy, but 16.7 in the isthmic tissue. In ovariectomized monkeys treated for 7 or 12 days with estradiol benzoate reciliation occurs, but to a significantly lesser extent in the fimbriae and ampullae than in the pre- or postovulatory animals; the degree of reciliation in the isthmus is not different from the values noted during the cycle. The ultrastructure of ciliated, deciliated and reciliating cells is described. Of much interest is the finding of cytoplasmic protrusions containing variable numbers of ciliary axonemal complexes. It is postulated that such internalization of ciliary micotubules may represent one way in which deciliation may be accomplished.

Sperm penetration into cervical mucus in vitro. II. Human spermatozoa in bovine mucus.

Human spermatozoa pentrate estrous bovine cervical mucus readily in vitro and maintain good motility and viability for a number of hours. They show pronounced unidirectional motion in mucus that has been aligned linearly. Data from tube preparations indicate that human spermatozoa from a given ejaculate travel more rapidly in estrous bovine mucus than in human midcyle mucus. They are prevented from penetrating luteal phase bovine mucus. The results are discussed in relation to a model of the molecular structure of cervical mucus, derived from laser light-scattering spectroscopy. In addition, it is suggested that bovine cervical mucus could be developed as a possible substitute for human cervical mucus in cases of infertility due to deficient endogenous mucus.

Sperm penetration into cervical mucus in vitro. I. Comparative studies.

Comparative studies have been carried out on the behavior of human and bovine spermatozoa toward homologous cervical mucus in vitro. In both cases the degree of sperm penetration and the pattern of sperm motility were influenced in a characteristic fashion by prior manipulation of the mucus: the most rapid and extensive penetration, and pronounced unidirectional motion, were seen in mucus that had been aligned linearly. By contrast, spermatozoa from rabbits, guinea pigs, rats, and mice were largely prevented from entering either midcycle human or estrous bovine cervical mucus, regardless of its physical arrangement. The observations on sperm motility patterns and the degree of penetration are discussed in relation to a model of the molecular arrangement of cervical mucus, derived in our laboratory from laser light-scattering spectroscopy.

A stochastic model for oviductal egg transport.

A mathematical description of ovum transport based on Langevin's diffusion equation is presented. The proposed model is deduced from qualitative features of this phenomenon, not induced from numerical fitting of experimental data. We demonstrate that egg transport in the ampulla of the rabbit oviduct can be represented as a one-dimensional random walk in a field of external force. The application of the model to describe isthmic ovum and sperm transport on the basis of simple random walk process is also discussed. The present formulation identifies and characterizes the forces involved in the motions of the ovum and predicts specific alternatives for physiological regulation of egg transport in the oviduct.

Laser light-scattering study of the effect of progesterone on sperm motility.

It is known that progestins can induce in the secretory cells of the cervix the excretion of a mucus that is highly viscuos, scanty, and impenetrable to spermatozoa. Mucus of this type is similar to that excreted during the luteal phase of the normal human menstrual cycle and the cow estrous cycle. It is a natural sequence to ask the question, do progestins also have a direct effect on sperm motility? With dynamic laser light-scattering we measured the motility of freshly washed human spermatozoa and of spermatozoa in the presence of a progesterone, both in terms of their swimming speed distribution as expressed in the spectrum of scattered light. The swimming speed was significantly reduced when the concentration of progesterone was three orders of magnitude greater than that of the physiologic level. This finding confirms the finding in earlier biochemical studies that progesterone has a distinct spermiostatic effect. We suggest this answer to the above question: progestin-releasing contraceptive devices may act on spermatozoa directly as well as in the secretory cells of the cervix.

PIP: Laser light scattering was used to determine the concentrations of progesterone that would inhibit sperm motility in vitro to judge whether the mechanism of progesterone action as a contraceptive was due to alterations of cervical mucus and, hence, sperm transport inhibition. 2 methods were used: one dissolved progesterone in a semen sample obtained by human masturbation, and one required implantation of a progesterone-containing silicone rod in cow uterine tissue in vitro. Swimming speed of spermatozoa was determined. From a control sample, it was found that the decay of sperm ability to swim was an exponential function of frequency. The swimming speed of washed sperm and treated sperm was measured by the spectrum of scattered light. Overall, the swimming speed was significantly reduced when the concentrations of progesterone were 3 orders of magnitude greater than the physiological level, confirming progesterone's spermiostatic effect. Apparently, progesterone acts on receptors of the sperm plasma membrane, causing sperm stasis. Since laser light scattering is noninvasive, it has a place in evaluations of sperm motility. This study showed that the concentration of progesterone can be as high as 2 mcg/ml in the cervix without causing an appreciable effect on sperm motility, but inhibition occurs at higher progesterone concentrations.

Comparative aspects of tubal anatomy and physiology as they relate to reconstructive procedures.

PIP: This article reviews the physiology of oviducts in many species, noting differences among various species of experimental animals, and complex mechanisms involved in gamete transport. Both of these topics, physiology and gamete transport, are related to their possible effects on reconstructive procedures of fallopian tubes. An understanding of the mammalian oviduct is essential before operative techniques for reanastomosis of tubes can be refined. The following topics are covered in this article: 1) the oviducts and supporting mesenteries; 2) the tunica serosa; 3) the muscularis; 4) blood and lymphatic vessels; 5) nerve supply; 6) fimbriae; 7) ciliogenesis in the oviductal epithelium, which is an estrogen-producing phenomenon; 8) oviductal ciliated epithelium and the direction of ciliary beat; 9) the ampulla of the oviduct and egg transport; 10) isthmoampullar junction and egg retention; 11) egg transport through the isthmus; and 12) sperm transport through the isthmus.^ieng

Proteolytic activity of guinea pig spermatozoa after induction of the acrosomal reaction in vitro.

After inducing the acrosomal reaction in guinea pig spermatozoa in vitro, the sperm were tested for proteolytic activity by applying them to membranes of fixed gelatin. One to 5% of them showed slight evidence of proteolytic activity, while the rest were completely negative. Sperm that had retained their acrosomes throughout the incubation period displayed intense proteolytic activity. These results suggest that proteinases may be lost from spermatozoa as a result of the acrosomal reaction.