Showing papers in "Biology of Reproduction in 1975"

Capacitation of Rabbit Spermatozoa in vitro

Abstract: This research was carried out to define in vitro conditions that would enable ejaculated rabbit sperm to fertilize ova in vitro. During 20 mm exposure of sperm cells to defined media of increasing NaCI concentrations, progressive removal or alteration of sperm surface seminal plasma antigenic components was initiated and completed as reflected by an immunological assay at approximately 300 and 380 mOsm/kg, respectively. Hypertonic (380 mOsm/kg) medium effected complete removal or alteration of these components as determined by the immunological assay during the first 5 mm of sperm treatment. Isotonic (305 mOsm/kg) treatment was shown to have effected removal or alteration of some but not all of the antigenic sperm coating seminal plasma components detectable by this means during 20 mm of exposure. Sperm within the perivitelline apace, presence of pronuclei. and 2- and 4-cell cleavage stages were taken as evidence for fertilization. None of 34 ova incubated for 27 h in vitro with once-washed sperm showed evidence of fertilization. Following treatment of sperm with hypertonic (380 mOsm/kg) medium prior to in vitro insemination, proportions of ova showing evidence of fertilization ranged from 8.1 percent (12 to 148 ova) to 72.2 percent (52 of 72 ova); and, following treatment of sperm with isotonic (305 mOsm/kg) medium, fertilization of ova ranged from 0 percent (0 to 19 ova) to 73.9 percent (SI of 69 ova) when results were considered according to individual male sperm donors. No large differences were found by the immunological assay that could be linked to variability of fertilizing ability of sperm from different bucks. Differences in metabolic characteristics of sperm cells was suggested as a likely reason for differences in fertilizing ability of sperm from different bucks. In experiments using sperm and ova from the same sources, no differences in fertilization results were apparent following 305 and 380 mOsm/kg sperm treatments, Initiation, but not completion, of removal or alteration of sperm surface seminal plasma antigenic components, then, was a necessary prerequisite to in vitro insemination for successful achievement of fertilization in vitro. Sperm penetration into ova occurred in some cases before 7-10 h after insemination (4 of 54 ova penetrated), but actively motile sperm were found within the perivitelline apace of other ova as late as 25 h after insemination. One hundred and seventy-six 2- and 4-cell stage embryos were transferred to 14 recipient does. Twenty-four embryos (13.1 percent) implanted in 6 recipients but most were resorbed. Three embryos resulting from in vitro fertilization with in vitro capacitated ejaculated rabbit sperm were carried successfully to term by 2 recipients and 2 of the offspring were males. This provides documentation for the authenticity of in vitro capacitation of ejaculated sperm of a mammalian species and represents the initial successful combination of in vitro sperm capacitation with in vitro fertilization and embryo transfer in the rabbit.

The Ovarian Cycle of the Bitch: Plasma Estrogen, LH and Progesterone

Abstract: Plasma levels of luteinizing hormone (LH) progesterone and estrogen were determined in pregnant and nonpregnant cycles of female beagle dogs. Mean basal LH levels during estrus and proestrus were 1.4 + or -.1 ng/ml and peaked at 7.5 + or -.8 ng/ml on the 1st day of estru s. Preovulatory increases in progesterone and LH appeared to be temporally correlated and were significantly elevated by the 2nd day of estrus (p less than .001). Progesterone levels reached their maximum on the 25th day of estrus (22.9 + or -2.7). Estrogen levles increased from 26 + or -4 on Day 10 prior to the LH peak to 62 + or -4 pg/ml 1 day before the LH peak. Once the LH peak was established estrogen levels fell rapidly. In the luteal phases of 12 pregnant and 10 nonpregnant cycles there were no marked differences in mean maximum progesterone levels the ranges of individual maximum levels or the time of their occurrence. After Day 30 progesterone levels declined gradually and were significantly (p less than .001) variable in nonpregnant cycles compared with pregnant cycles once progesterone levels dropped below 1 ng/ml. Progesterone levels showed significant (p less than .05) daily changes during the 2 days prior to parturition. Plasma Estrogen levels were constant in nonpregnant cycles but significantly (p less than .01) increased during the 3 weeks prior to parturition. The results suggest that plasma estrogen initiates or potentiates the preovulatory release of LH that the early preovulatory release of progesterone may encourage the LH peak surge and the onset of behavioral estrus and that mechanisms exist that maintain elevated levels of plasma progesterone throughout pregnancy.

The Relationship Between Sperm Concentration and Fertilization in vitro of Mouse Eggs

Abstract: A 300 fold range of epididymal sperm concentrations (0.3-90 X 105/ml) has been tested to determine whether there is a relationship between sperm concentration and level of fertilization achieved in vitro of mouse eggs. While very low concentrations (0.3-1.25 X 105/ml) resulted in relatively low fertilization (43-64 percent), those in the range of 2.5-90 X 105/ml gave fertilization rates of 80- 94 percent. Consistently high results were obtained with sperm counts

Absence of Arms in the Axoneme of Immobile Human Spermatozoa

Abstract: The spermiograms of an infertile man were unique in showing normal parameters except for the presence of total immotility in 100 per cent of the spermatozoa. In 100 per Cent of the axonemes of such spermatozoa there was a consistent lack of arms on the doublets of the axoneme. The same feature was found in all stages of spermatid formation. Other less consistent abnormalities were the occasional appearance of extra coarse fibers and axonemal microtubules, and an abnormal relationship between the longitudinal columns of the fibrous sheath and the doublets of the axoneme. All other ultrastructural details compared well with normal spermatozoa. Detailed descriptions of the substructural organization of axonemes from a wide variety of sources have to some extent helped to clarify our understanding of ciliary and flagellar motility. However, at the present time significant information at the molecular level is needed before a functional model comparable to the one known for muscle contraction can be achieved. The presence of a rather unique axonemal defect in the spermatozoa of a sterile man may help to throw some light on these problems. In this report the history of such a patient and the ultrastructural findings in his spermatozoa will be described.

The Glutathione and Thiol Content of Mammalian Spermatozoa and Seminal Plasma

Abstract: The glutathione and thiol contents of spermatozoa and seminal plasma of several animal species have been examined. Washed, ejaculated or epididymal spermatozoa from man, dog. ram and goat were found to contain about 5 (I - 13) nmoles of glutathione per 10’ cells. Little or none was present in rabbit and boar spermatozoa. In contrast, the total thiol content of the sperm samples was 350 (95-700) nmoles/I0’ cells, but almost all of it was protein-bound. Dog and human seminal plasma similarly contained thiol groups which were almost entirely protein-bound. The concentration of glutathione in seminal plasma was less than 2 tiM. The spermatozoa which contained glutathione also exhibited substantial glutathione reductase and glutathione peroxidase activities. These findings suggest the possibility that these enzymes and glutathione may be functional in the spermatozoa of some animals as a protective mechanism against oxidative damage by H2O2. as has been demonstrated in other cells.

Circulating levels of steroids and chorionic gonadotropin during pregnancy in the rhesus monkey, with special attention to the rescue of the corpus luteum in early pregnancy.

Abstract: Circulating levels of progesterone and estrogen in the pregnant rhesus monkey are indistinguishable from those of the nonpregnant animal until 12 days after the LH peak, when the first significant rise in chorionic gonadotropin (RhCG) levels is noted concomitant with an abrupt elevation in progesterone levels (the corpus Iuteum rescue). Serum RhCG concentration rises rapidly (doubling time = 2 days) to a peak at about day 23 of pregnancy, then falls to undetectable levels at day 35, and remains undetectable throughout the rest of gestation. Serum estrone and estradiol levels rise and fall with RhCG titers in early pregnancy, reaching a nadir on day 35 of gestation before placental estrogen secretion (largely estradiol) supervenes. Circulating estrogens reach a high and variable plateau at about day 80 of pregnancy and rise to maxima on the day prior to parturition. Serum progesterone levels in early pregnancy, while rising abruptly at the outset of RhCG secretion, fall in the face of waxing RhCG titers and increase again only as RhCG levels fall. Ovariectomy on day 23 of pregnancy causes a sharp fall in circulating levels of estrogens and no compromise of the continuation of pregnancy. These data suggest that the order of hormone production by the placenta is RhCG. first discernable at about 10-I I days after ovulation, followed by progesterone, whose secretion by the placenta is initiated prior to day 23 of pregnancy since ovariectomy at this time causes no fall in circulating progesterone levels, and lastly, estradiol and estrone. which rise rapidly in peripheral serum after day 35 of pregnancy. These data support the hypothesis that the shortlived stimulation of progesterone secretion by the corpus luteum in early pregnancy is caused by the initiation of placental RhCG secretion and that the failure of Iuteal progesterone secretion to be maintained in the face of rising RhCG titers may be an expression of the luteolytic effect of the increased luteal estrogen production also stimulated by this gonadotropin.

The Peritubular Tissue in the Normal and Pathological Human Testis. An Ultrastructural Study

Abstract: The peritubular tissue of the human testis has been examined by light and electron microscopy in biopsies from 5 normal men, 10 untreated men with hypogonadotrophic hypogonadism and 52 men with oligospermia or azoospermia. In the normal, the lamina propria of the tubules consisted of two alternating layers of collagen fibres and myoid cells to whose surface a thin layer of basement membrane-like material and microfibrils was closely applied. In hypogonadotrophic hypogonadism, the immature seminiferous cords were surrounded by less well defined layers of myoid cells and collagen fibres and the myoid cell surfaces did not exhibit the coating of basement membrane-like material and microfibrils. In the patients with azoospermia or oligospermia. three patterns of organization of the lamina propria were apparent: (I) The normal appearance. (2) Increase in thickness of the layers of collagen fibres. (3) Disorganization of the lamina propria by a marked increase in the thickness of either the basement membrane-like material or microfibrillar layers immediately adjacent to the myoid cells. The third pattern was found in patients with severely damaged testes such as in seminiferous tubule hyalinization. The observations indicate that the thickening of the lamina propria of the seminiferous tubules seen in some damaged testes does not involve the appearance of new components but widening of existing structures. It is generally agreed that the myoid cells,

The Effect of Estrogen and Progesterone on Uterine Prostaglandin Biosynthesis in the Ovariectomized Rat

Abstract: The role of 17a-estradiol and progesterone in the in vivo synthesis of prostaglandins by the ovariectomized rat uterus was investigated. Seven days after ovariectomy, rats were treated with progesterone (2 mg X 2 days) then given a single injection of estradiol. Prostaglandin E (PGE) and F (PGF) were determined by RIA in uterine tissue and uterine vein plasma (UVP) at 0, 0.5, 1, 2, 4, 8, 12 and 24 h after the administration of 1713-estradiol (10 Mg). PGF and PGE content (ng/uterus) remains constant from the time of estradiol administration to the latest time period studied. PGF and PGE concentrations (ng/100 mg uterine wt) show a gradual decline during the course of estrogen action which becomes significant only 12 and 24 h after estradiol administration and is correlated with the increase in uterine weight. Following progesterone administration, there is a significant increase in both uterine PGE content and concentration from ovariectomized levels. These results indicate that levels measured in UVP represent a true de novo synthesis of prostaglandins and could not be accounted for by the release of stored material into the uterine vein. PGF levels in UVP rose from control values of 2.42 ± 0.53 ng/ml to a maximum of 23.30 ± 4.96 ng/ml at 12 h and by 24 hhad returned to basal levels. A comparison with animals not treated with progesterone revealed low levels of PGF in UVP at all time periods studied (0.5-12 h). PGE levels in UVP after estradiol administration to progesterone-treated and untreated rats were similar, although at 12 h, levels in progesterone-treated rats were significantly greater than the untreated group. Determinations of estradiol in peripheral plasma by RIA revealed a sharp peak 1 h after administration followed by a rapid decline to control values by 12 h. Using the optimal conditions for prostaglandin biosynthesis as determined in the first part of this experiment (i.e.: 2 days of progesterone pretreatment and uterine cannulation 12 h after estradiol administration), the effect of estradiol doses between 0.001 and 100 g was investigated. Maximum UVP levels of PGE and PGF were obtained with doses greater than 1.0 Mg estradiol. The simultaneous administration of progesterone (2, 10 or 50 mg) with estradiol (10 Mg) significantly reduced PGF concentration in UVP, whereas only 50 mg progesterone was effective in significantly reducing PGE levels. Fifty mg of progesterone alone was without effect on PGE and PGF levels in UVP. It is concluded that estrogens are the predominant ovarian steroids involved in the regulation of uterine prostaglandin biosynthesis in the rat; however, important roles for progesterone in the expression of estradiol stimulated activity are indicated.

Proacrosin from Rabbit Epididymal Spermatozoa: Partial Purification and Initial Biochemical Characterization

Abstract: Proacrosin, the zymogen form of acrosin was extractedwith 0.25N H2SO4 from unwashed rabbit cauda epididymal spermatozoa. Sephadex G-75 column chromatography of the extract resolved two peaks of epididymal sperm proacrosin, both of which contained low amounts of acrosin prior to activation (a total of 5 percent of the final acrosin activity obtained from complete activation of proacrosin), and were free of trypsin inhibitor. The peak eluted first (peak I) represented 85 percent of the total proacrosin and had an apparent molecular weight (MW) of 73,000 as determined by Sephadex G-100 column chromatography. Complete “autoactivation” of sperm proacrosin peak I resulted in the production of an acrosin with a MW of 38,000. Proacrosin was alsofound in washed epididymalsperm uncontaminated by free cytoplasmic droplets or epididymal fluid. Sperm proacrosin “autoactivation” appeared to be a second order autocatalytic process in which the product of the activation accelerated its own formation. The “autoactivation” was stimulated by Ca�� and inhibited by � These properties of sperm proacrosin agree with our previous data from studies of rabbit testis proacrosin. Also the acrosin obtained by “autoactivating” the sperm and testis proacrosins, had similar apparent Km values for BANA (7.8-10.5 X 10-5M), BAEE (2.1-4.3 X 105M) and TAME (2.4-3.3 X 10-SM), similar pH optima (pH 8.2-8.4), and were similarly inhibited by several trypsininhibitors. These resultssuggeststrongly that rabbit epididymal sperm and rabbit testis proacrosin are identical proteins. Conversion of sperm proacrosin to acrosin (involving the hydrolysis of at least one peptide bond)would appeartobe necessaryifacrosinplaysaroleinfertilization.

Effect of 5α Reduced Androgens on Sex Accessory Organs. Initiation and Maintenance of Spermatogenesis in the Rat

Abstract: The effect of several androgens on initiation and maintenance of spermatogeneSis was studied in newborn estrogen-blocked and in adult hypophysectomized rats. The steroid treatment inhibited testicular and body weight in all animals. Treatment of estrogen-blocked animals with androgens resulted in a stimulation of the growth of sex accessory organs, except in the case of androsterone. Initiation of spermatogenesis and its progression to mid pachytene stage (first wave) was observed in all steroid treated rats. In estrogen-blocked animals the first wave of spermatogenesis proceeded only to pachytene spermatocytes. Administration of testosterone propionate. dihydrotestosterone or 5a-androstanediol to these animals resulted in completion of meiosis and formation of spermatids up to Step 8 of spermiogenesis. Completion of meiosis was not observed in androsterone treated rats. In hypophysectomized animals all 5a-reduced androgens maintained spermatogenesis similar to testosterone.

Gene expression and macromolecular synthesis during preimplantation embryonic development.

Abstract: The most extensively studied systems of early embryonic development are ones far removed from man and other mammals and include, most notably, the echinoderms and amphibia. Based on work with these and other organisms, there has been built-up an overall picture in which much, if not all, of early post-fertilization development is controlled by messenger RNAs (mRNAs) which are synthesized in the egg but are not activated until after fertilization has occurred (Gross, 1967; Davidson, 1968; Brown and Dawid, 1969; Brachet and Malpoix, 1971). The presence and activity of such masked messenger RNAs (mmRNAs) were originally defined in experiments in which early development was shown to be invulnerable to the effects of actinomycin D, an inhibitor of RNA synthesis. However, the physical existence of these mmRNAs now seems to be firmly established, and it has been possible to isolate and use them to govern protein synthesis in vitro (Gross, et al. 1973; Skoultchi and Gross, 1973). With the development of techniques for the biochemical analysis of the mammalian embryo, it is natural that the question of how early mammalian development is controlled should be of great interest. While the presence or absence of mammalian mmRNAs has not as yet been established, the available information indicates that all stages of preimplantation mammalian development are affected by the genetic activity of the embryo itself. It will be the purpose of this paper to summarize the relevant information obtained principally from our own studies on the preimplantation mouse embryo and, when applicable, from the studies of others with mouse and rabbit embryos. Unless otherwise stated, the material will refer to the developing mouse embryo. between fertilization and implantation. Recent reviews of this subject have been published by Wolf and Engel (1972), Fowler and Edwards (1973), and Biggers and Stern (1973). The evidence will be reviewed in order from the least to the most direct, and the major point to be considered will be the role of embryonic gene action in governing early mammalian development. However, although our emphasis is on genetic control, it is well to keep in mind that gene activity by itself is unlikely to be the whole explanation of development and differentiation. This point was well stated in a recent book by Wright (1973). “Application of the words cause’ or trigger’ to particular components within a differentiating system may delude the user into feeling that there exist certain ‘spontaneous’ or independent factors uniquely capable of causing differentiation to occur. . . . There is no a priori reason for singling out any particular biochemical event on which differentiation depends as being more essential or necessary than others. ... Such an approach renders an objective analysis difficult and stresses the role of components which may or may not be limiting the rate of the differentiation process in question. It appears to be no more justilied or useful to choose selective gene activation as ‘the’ basis of differentiation than to choose, for example, substrate availability.”

Ovarian Progesterone Levels During in vitro Oocyte Maturation and Ovulation in Xenopus laevis

Abstract: Endogenous progesterone was measured in ovaries of the frog Xenopus laevis following the administration of gonadotropins that induce meiotic maturation and ovulation of large oocytes. Ovarian pieces were incubated for 0, 1, 3, 5, or 10 h in Gurdon’s solution in the presence or absence of human chorionic gonadotropin (HCG; 20 lU/mI) or a frog pituitary homogenate (FPH; 0.04 pituitary/mI). Each incubation sample was scored for ovulation and maturation at the end of its incubation period, homogenized in the medium, and extracted for progesterone with petroleum ether. Following purification on Sephadex LH-20, extracts were assayed for progesterone by a radioimmunoassay that was validated for use with frog ovarian tissue. In 13 experiments, mean (! SEM) progesterone concentration in untreated ovarian tissue was 3.7 ± 0.5 ng/gm at time zero and progesterone levels did not change significantly during 10 h of incubation. HCG-treated tissue (n = 6) exhibited a linear, two-fold increase in progesterone for the first 3 h of incubation and maintained that level for the remainder of the incubation. FPH treatment (n = 10) produced a linear six-fold increase in progesterone over the course of 10 h. Mean progesterone content across time was significantly greater (P<0.005) in FPH-treated tissue than in HCG-treated tissue. Both differed significantly (P<0.05) from untreated controls. In tissue treated with HCG the percent oocyte maturation was correlated (P<0.10) with both mean progesterone concentration across time and progesterone concentration at 10 h of incubation; ovulation did not occur. In FPH-treated tissue progesterone concentration was not correlated with percent maturation but was correlated (P<0.05) with the number of ovulations/gm. Treatment of ovarian pieces with the gonadotropins following a 10 h incubation in Gurdon’s solution produced effects similar to those following treatment at time zero. In vivo treatment with HCG prior to in vitro incubation of ovarian tissue with HCG or FPH resulted in an eight- or eleven-fold increase, respectively, in peak progesterone concentration as compared to tissue that was not pretreated in vivo. In two experiments large, preovulatory follicles contained sufficiently more (P<0.05) progesterone following FPH treatment than did smaller follicles. Both large and small FPH-treated follicles had significantly higher levels of progesterone than untreated control tissue.

Acceleration of the Acrosome Reaction and Activation of Guinea Pig Spermatozoa by Detergents and Other Reagents

Abstract: In vivo, guinea pig spermatozoa do not begin to undergo the acrosome reaction and to penetrate eggs until 4 to 10 h after they are deposited in the female genital tract. When spermatozoa are incubated in a modified Krebs-Ringer’s solution containing 0.003 percent Hyamine, the majority of them exhibit both the acrosome reaction and activation within 10-15 mm and become capable of penetrating eggs. The acrosome reaction and activation induced by Hyamine are reversibly blocked when calcium ions are omitted from the medium. Other reagents capable of inducing an accelerated acrosome reaction and/or activation include: Triton, Brig, saponin, nonanol, nystatin, mersalyl acid, dibucane, procaine, neotetrazolium chloride, nitro blue tetrazolium, chloroquine, calcium ionophore, unheated sera of the rabbit and guinea pig, rabbit complement and pronase. The study shows that guinea pig spermatozoa are potentially capable of an immediate acrosome reaction and activation upon leaving the epididymis.

Uterine Secretion in Mammals: Synthesis and Placental Transport of a Purple Acid Phosphatase in Pigs

Abstract: A fluorescent antibody technique was used to study the site of synthesis, movement and steroid control of porcine purple acid phosphatase uterine protein in uterine and oviducal tissues on Days 0, 3, 6, 9, 12, 15 and 18 of the estrous cycle. Uterine, oviduct and placental tissues were obtained from pregnant gilts on Days 9, 15, 18, 20, 30, 50, 70 and 90 of gestation. In addition, uterine tissue was obtained from ovariectomized animals subjected to various steroid hormone treatments. The oviducal eoithelium and uterine surface and glandular epithelium showed various degrees of fluorescence at different stages of the estrous cycle with an apparent change in pattern coincidental with fluctuations in ovarian steroid levels. It was concluded that estrogen may be required for initiation of synthesis of this protein, but progesterone induced full synthesis and secretory activity. Enzyme assays performed on uterine flushings from each day of the estrous cycle support the immunofluorescent results. In pregnant gilts, oviducal and uterine surface and glandular epithelium showed fluorescence at all stages of gestation studied. A significant difference was observed in the pattern of fluorescence between uterine endometrium of early pregnancy and that of the corresponding stages of estrous cycle. As pregnancy progressed, placental areolae became strongly fluorescent, indicating absorption of uterine gland secretions by these structures. Inhibition of uterine secretory activity took place in association with rising estrogen levels toward the end of gestation. This study suggests that the purple acid phosphatase is synthesized and secreted by the uterine epithelial cells under the influence of progesterone and that placental areolae serve as sites of absorption and transport of this protein across the chorio-allantoic membranes and into the allantoic fluid where it is sequestered during pregnancy.

Endocrine Changes in the Pig During Late Pregnancy, Parturition and Lactation

Abstract: Peripheral plasmal levels of progestins. corticosteroids. and estrogens were determined in gilts beginning approximately 2 weeks prior to and at various intervals during parturition. as well as for up to 37 days postpartum in Yorkshire-Hampshire cross pigs. In addition, the same steroids were determined in 2 animals which aborted due to leptospirosis infection. Blood was obtained via a catheter chronically implanted in the lesser saphenous vein. Progestins declined slowly in late gestation from about IS ng/ml plasma until about 2 days prior to parturition. when levels decreased rapidly to about 3-4 ng/ml at delivery. A further decline in progestins was noted within 24 h after delivery with levels remaining low for up to 37 days postpartum. Progestin levels did not change during delivery. Corticosteroids. although variable in concentration, increased significantly 24 h prior to and during parturition. Estrogens increased continuously from II days prepartum (2 ng/mI) to 3 days prepartum (6.4 ng/ml), remaining constant through parturition with a precipitious decline observed by 24 h postpartum (2.5 ng/ml). Levels remained low (0.2 ng/mI) for up to 37 days after delivery. In the 2 animals that aborted, progestin and corticosteroid patterns were somewhat similar to those noted for animals undergoing normal delivery, while estrogen levels differed from normal patterns in that estrogens declined in one animal and were low with a slight increase in the other animal prior to delivery. Although the number of observations is limited, it appears that premature delivery in the pig during late gestation due to disease processes is not accompanied by completely normal endocrine preparatory changes (estrogens in particular). This may be due to the fact that while the female may be able to respond in a normal manner to premature induction of labor, porcine fetuses appear to be immunologically incompetent (as compared to bovine and ovine fetuses) with fetal death the main result of exposure to stressor agents. Thus delivery may be preceded by fetal death which does not allow for the initiation of normal endocrine patterns. Endocrine changes occurring during the onset of parturition have been studied in various mammalian species. The decline in peripheral plasma levels of progesterone in most species, e.g. rat, rabbit, hamster, goat, ewe and cow (see Davies and Ryan, 1972; Thorburn et a!., 1972 for references), regardless of an ovarian or placental origin, seems to be an important prelude to parturition. An exception is seen in humans (Llaur#{243}, et a!., 1968; Yannone, et a!., 1968) and subhuman primates (Stabenfeldt and Hendrickx, 1973)

The ultrastructure of the canine testicular interstitial tissue.

Abstract: The seminiferous tubules of the dog testis are surrounded by a boundary tissue composed of a basal lamina and a layer of peritubular cells that is from one to six cells thick. Myoid cells, arranged in a layer only one cell thick. are the innermost cells of the boundary tissue. These cells are characterized by the presence of numerous microfilaments 5 nm in diameter. micropinocytotic vesicles, intracellular densities near the plasma membrane, and a basal lamina on both faces. In the interstitial tissue, clusters of Leydig cells are enveloped by a basal lamina. and the individual cells are separated by a varying intercellular space. Leydig cells are joined by gap junctions, septate-like junctions, and rudimentary desmosomes. Within their cytoplasm are many 5-nm wide microfilaments. The smooth endoplasmic reticulum is seen either as tubules or simple cisternae separated by microfilaments in the less developed Leydig cells and as extensive whorls of fenestrated smooth endoplasmic reticulum surrounding lipid droplets or mitochondria in the larger Leydig cells. Another cell type is present in the dog testis that has many of the cytological characteristics of the Leydig cell. This cell, however, is located within the boundary tissue of the seminiferous tubules between the myoid cell layer and the lymphatic endothelium. These Leydig-like cells may represent a stage in Leydig cell differentiation. The testicular lymphatics occur both as vessels and as sinusoids and often separate the boundary tissue from the interstitial tissue.

Sperm Binding Characteristics of the Murine Zona Pellucida

Abstract: Experimental conditions were established for an investigation of the sperm binding characteristics of the mouse zona pellucida. Fresh epididymal sperm binding to zonae of unfertilized eggs was a time-dependent process, reflecting the occurrence of sperm capacitation under in vitro incubation conditions. In contrast, capacitated sperm binding in large numbers to zonae of unfertilized ova occurred independently of temporal considerations. A dramatic difference was apparent in the binding of capacitated sperm to zonae of unfertilized eggs (>100 per zona) and to in vivo produced 2-cell embryos (0-5 per zona), providing a basis for assessing the occurrence of a zona reaction. Under comparable conditions, capacitated sperm bound in large numbers to zonae of in vitro produced 2-cell embryos. Possible reasons for a subnormal or inadequate zona reaction in eggs inseminated in vitro have been discussed.

Diurnal Rhythms in Gonadotropins and Progesterone in Lactating and Photoperiod Induced Acyclic Hamsters

Abstract: Levels of LH, FSH, and progesterone in serum were measured in lactating hamsters and in hamsters in which acyclicity was induced with altered photoperiod. Lactating hamsters were found to have low titers of LH, FSH, and progesterone in serum at 0900 (lights on 0500-1900) on Days 4, 9, 14, and 19 of lactation and increased levels of these hormones at 1600. Levels of LU and FSH in serum at both 0900 and 1600 remained relatively constant throughout lactation. In contrast, levels of progesterone in serum obtained at both 0900 and 1600 sampling times increased as lactation progressed. Ovariectomy on Day 9 of lactation reduced serum levels of progesterone at both 0900 and 1600 and eliminated the afternoon surge in progesterone in animals bled 5 days after surgery. The levels and pattern of LH in serum remained unchanged after ovariectomy in lactating hamsters. However, serum FSH levels in the ovariectomized, lactating animals were elevated at both 0900 and 1600 when compared to levels present in intact, lactating hamsters bled at the same times. Females which were acyclic due to altered photoperiod displayed similar patterns of LH, FSH, and progesterone in serum. Levels of LH, FSH, and progesterone in serum were low at 1000 (lights on 0500-1500) and were increased 2 to 10 fold at 1500. Ovariectomy was followed by lower progesterone levels in serum at 1000 and 1500 and eliminated the afternoon rise of this hormone. Serum levels of LH were unaffected by ovariectomy. As in lactating hamsters, levels of FSH in serum were elevated 3-4 days following ovariectomy at both bleeding times, but the levels were higher at 1500. These results indicate that acyclicity induced by lactation or exposure to a short photoperiod is characterized by similar diurnal patterns of circulating hormones in the hamster.