Bela J. Gulyas

Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: Effects on fertilization and early development

During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.

In vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: progesterone production by dispersed luteal cells

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term in vitro studies of progesterone production by suspensions of collagenase-dispersed luteal cells in the presence and absence of exogenous gonadotropin (human chorionic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the incubation medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion significantly above basal level (63.7 +/- 13.1 versus 24.7 +/- 5.5 ng progesterone/ml/5 x 10(4) cells/3 hr, X +/- S.E., n =6 ; p less than 0.05). However, luteal cells from early pregnancy (23-26 days after fertilization) secreted siginificantly less progesterone than cells of the non-fertile menstrual cycle (3.6 +/- 2.4 versus 24.7 +/- 5.5 ng/ml/5 x 10(4) cells/3 hr, n =3 ; p less than 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108-118 days gestation ) luteal cells exhibited partially renewed function, and near the time of parturition (163-166 days gestation) basal and HCG-stimulated progesterone secretion (30.2 +/- 5.6 and 63.0 +/- 13.0 ng/ml/5 x 10(4) cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when steroidogenic activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitive steroidogenesis similar to that the corpus luteum of the menstrual cycle.

Identification of mammalian sperm surface antigens: II. Characterization of an acrosomal cap protein and a tail protein using monoclonal anti-mouse sperm antibodies.

Monoclonal anti-mouse sperm antibodies have been produced by fusing mouse myeloma cells with spleen cells from rats immunized with epididymal sperm of C3H mice, Immunoprecipitation and immunoperoxidase techniques showed that one such monoclonal antibody, AMS IV-33, recognized a 200 000 dalton protein localized on the acrosomal cap of the sperm cell. Two other monoclonal antibodies AMS IV-54 and -76, reacted with a 68 000 dalton component on the surface of the sperm tail. Both antigenic targets were species specific and were present in about equal amounts on sperm from several different strains of mice. The tail protein was sperm specific, whereas the antibody reacting with the acrosomal cap protein also appeared to react somewhat with antigens present in other mouse tissues.

Role of the primate placenta in cortisol secretion by the maternal adrenals

Peripheral serum cortisol levels were measured throughout gestation in 5 intact pregnant rhesus monkeys (Macaca mulatta) and 3 hypophysectomized-fetectomized monkeys, leaving the placentas in situ and viable. These monkeys, as well as 4 other groups used to separately control for effects of pregnancy, hypophysectomy, or fetectomy, were unilaterally (left) adrenalectomized to permit comparisons of adrenal gland weights. Circulating cortisol levels of intact pregnant monkeys tended to rise slightly with advancing gestation. However, hypophysectomy at 70 to 73 days after fertilization caused a marked decline (p < 0.01) in serum cortisol concentrations to about 1/2 the preoperative level. These monkeys were fetectomized at 107 to 114 days without further reduction in circulating cortisol levels. In hypophysectomized-fetectomized monkeys, either surgical removal of the placentas near term or abortion was followed by a rapid decrease in peripheral cortisol to undetectable concentrations. Their cortisol levels were 5 to 12 times higher in left adrenal venous effluent than in peripheral circulation on the day of placental delivery. The presence of a viable placenta protected against the extensive adrenocortical involution seen in nonpregnant hypophysectomized monkeys (p < 0.01). Fetectomy, alone or in combination with hypophysectomy, did not alter left adrenal gland weights from those of intact pregnant monkeys. Thus, continued cortisol secretion and maintenance of adrenal weight in hypophysectomized-fetectomized monkeys, in the presence of a functional placenta, supports the existence of a placental adrenocorticotropin in this primate.

Maternal estrogen and progesterone levels after hypophysectomy in early pregnancy and in term fetuses or newborn monkeys

Pregnant rhesus monkeys (Macaca mulatta) were hypophysectomized at 8-10 weeks gestation to determine effects on plasma levels of estrone (E1), estradiol-17beta (E2), and progesterone (p). The first group of monkeys was subsequently fetectomized at 107-114 days. After hypophysectomy there was an initial decrease in maternal peripheral plasma E2 followed by a rise to preoperative levels within 4-5 weeks. Plasma levels of E1 and P were not markedly altered. After fetectomy, peripheral estrogen concentrations, especially E2, declined markedly. In the second experimental series, we have examined the effects of maternal hypophysectomy on levels of E1, E2 and P either (1) in both mother and newborn baby or (2) in mother, term fetus and umbilical vein. Groups of hypophysectomized and intact pregnant monkeys (3 each) were delivered by cesarean section at the expected time of parturition. Other hypophysectomized and intact monkeys (2 each) delivered normally. E2 levels were elevated significantly in plasma of hypophysectomized monkeys at the time of cesarean delivery and in newborn babies of hypophysectomized mothers shortly after parturition. Escept for these differences, the maternal hypophysis apparently is not a major factor in the control of E1, E2 and P concentrations in pregnant rhesus monkeys.

Fine structure of the luteal tissue

The corpus luteum is unique among endocrine organs, because it has a transient existence, whether it be short lived — as in case of the extrous and menstrual cycles, or long lived — as in case of pregnancy and pseudopregnancy. Furthermore, the gradual demise of one population of organ(s) heralds the potential for the development of a new set of similar organ(s). Yet, through its secretions the corpus luteum exerts a major influence on the reproductive function of the individual during its lifespan.

Association of microperoxisomes with the endoplasmic reticulum in the granulosa lutein cells of the rhesus monkey (Macaca mulatta)

SummaryAldehyde fixed tissue from monkey (Macaca mulatta) corpus luteum was incubated in alkaline 3,3′-diaminobenzidine (DAB), and prepared for electron microscopic histochemical observations. The association of microperoxisomes with the granular (GER) or agranular (AER) endoplasmic reticulum was reconstructed from serially sectioned tissues and by tilting of specimens in the microscope. Out of 107 microperoxisomes, 106 were directly associated with the AER. Two different forms of attachment were found between microperoxisomes and the AER. In the first type of connection, the lumen of the AER and that of the microperoxisome are confluent. In the second, ungulate type of connection, a blunt-end structure either is inserted into an invagination of the AER, or penetrates into the lumen of the AER. The lumen of the lingula is confluent with the microperoxisome, but not with the AER. In addition to these connections, fine thread-like structures were observed extending between AER and adjacent microperoxisomes.

The dependence of annulate lamellae formation on the nucleus in parthenogenetic rabbit eggs

SummaryPreviously it has been shown that, in the rabbit, although annulate lamellae (AL) are absent in the follicular oocytes, they appear in the fertilized eggs after the formation of the pronuclei. Furthermore, neither pronuclei nor AL appear when unfertilized eggs are aged in vivo or in vitro. This study was undertaken to determine whether AL formation requires presence of an intact nucleus, or whether the sperm alone contains the stimulatory factors essential to AL synthesis. Rabbit eggs were exposed to 10°C, then incubated for 24 hours. Control eggs were incubated without cold-treatment. Electron microscopic observations indicated that two-thirds of the eggs formed one or two ‘pronuclei,’ or subnuclei. The remainder one-third of the cold-treated eggs and the control eggs failed to form ‘pronuclei.’ AL were present in large amounts only in those activated eggs (parthenogenones) which formed ‘pronuclei.’ AL were absent in the control and the non-activated experimental eggs, both of which failed to form a ‘pronucleus.’ A few small AL were observed in eggs with subnuclei. Condensed fine textured nucleoli appeared precociously during cold-treatment in some eggs and they were present in the ‘pronuclei’ of activated eggs. It was concluded that the sperm is not necessary for AL formation, but the presence of an intact nucleus is mandatory.

Progesterone production by dispersed monkey (macaca mulatta) luteal cells after exposure to trypsin

In an attempt to justify use of trypsin to achieve more thorough dispersion of luteal cell clumps in vitro, progesterone (P) production by collagenase dispersed monkey luteal cells from the mid-luteal phase corpus luteum (CL) was examined in vitro either after 10 min, or continuous (3h) exposure to trypsin (TR). In the first experiment, cells were pre-incubated in TR, then incubated at 37 degrees C for 3h with human chorionic gonadotropin (hCG) after the addition of soybean-trypsin inhibitor (STI). Pre-incubation of luteal cells with TR had no effect on the level of P production under basal conditions. Cells that were preincubated with TR responded to hCG stimulation with increased progesterone secretion (P less than 0.01) in a fashion similar to untreated cells. P production in response to hCG was independent of TR concentration over the range of 0.05% to 0.2% during the pre-incubation period. However, continuous exposure (3h) of cells to TR significantly depressed (P less than 0.01) basal P secretion and inhibited the response to hCG. We conclude that TR had no effect on the biopotency of hCG per se, but probably the over-exposure to TR had an adverse effect on the LH/hCG receptors. Addition of STI after a 10 min pre-incubation with TR, prevented these deliterious effects, thereby permitting the use of TR to improve the completeness of luteal cell dissociation.

Identification of mammalian sperm surface antigens. I. Production of monoclonal anti-mouse sperm antibodies**Preliminary observations of this study were presented at the Thirty-Seventh Annual Meeting of The American Fertility Society, March 14 to 18, 1981, Atlanta, Georgia, and received the Associate Members’ Award.

Surface antigens of mammalian sperm were studied by use of monoclonal antibodies (MAs). Six hybridoma cell lines were obtained by fusion of mouse myeloma cells with spleen cells from rats immunized with unwashed, epididymal sperm from C3H mice. Quantitative assessment of antibody binding, using a solid phase, antibody-protein A assay, indicated that four MAs bound to integral, sperm surface antigens; two others bound to nonintegral sperm antigens or epididymal fluid components. Immunofluorescence studies showed specific binding of individual MAs to localized regions: acrosome, midpiece, and midpiece and tail. All of these MAs inhibited sperm-egg binding, and those to the midpiece and/or tail immobilized sperm cells. The monoclonal antibodies provide probes for immunochemical characterization of sperm antigens and for elucidation of the role of the antigens in sperm.