In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen

Summary In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S−F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors—the characteristic of fetal Leydig cells. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This work was supported by Contract NO1-CB-33907, and Grants No. 1 RO1 AG00042 and No. 5 RO1 CA16952 from the National Institutes of Health.     

Ascaris suum and Toxocara canis: Egg extract antigens in guinea pigs and the macrophage migration inhibition test

Guinea pigs immunized with 500 μg of either Ascaris suum or Toxocara canis egg extracts, emulsified with Freund's complete adjuvant, were skin tested with both the homologous and heterologous antigens. Cross-reactions were observed in both groups. Migration of macrophages from sensitized animals was more inhibited by homologous than by heterologous antigens. Lymph-node lymphocytes from sensitized animals were stimulated to incorporate [⁸H]thymidine similarly by both antigens.     

Appearance and endocytic activity of epithelial cells from human efferent ducts in primary monolayer culture

Summary The culture of epithelial cells lining human efferent ducts, obtained from prostatic carcinoma patients, is described. Ciliated cells were observed to beat for at least one month on plastic. On pervious filters low cuboidal cells characterized the monolayers. Cells comprising monolayers over the filter were 5 to 9 m in height whereas taller cells were found over the original fragments (14 m). Some non-ciliated cells contained dark and light vacuoles, others were found to lack them. Both non-ciliated and ciliated cells maintained tight junctional complexes restricting the paracellular movement of horseradish peroxidase. Both types of cultured cells exhibited fluid-phase and adsorptive endocytosis from both apical and basal surfaces. It is reported for the first time that the monolayers form high resistance barriers (150 cm²) that prevent the apical medium from draining to the basal compartment over 24 h.     

Protein phosphorylation is essential for formation of male pronucleus in bovine oocytes

This study examined the association between the morphological and protein phosphorylation events following sperm penetration of in vitro matured and in vitro fertilized bovine oocytes. Oocytes were labeled with [³²P]‐orthophosphate at 3 hr intervals from 3 to 18 hr of following insemination. The phosphorylation of protein complexes of 23 kDa and 18 kDa specifically increased with the formation of male and female pronuclei. In addition, oocytes were treated with 6‐Dimethylaminopurine (6‐DMAP) or Okadaic acid (OA) at 0, 3, 6, and 9 hr respectively following insemination. Although the formation of female pronucleus was not affected by 6‐DMAP, the male pronuclear formation was completely inhibited by the presence of 6‐DMAP at 0 and 3 hr of post insemination. The formation of both pronuclei was inhibited by the presence of OA at any time following insemination. These results suggest that the male pronuclear formation is associated with protein phosphorylation and that the formation of the male and the female pronuclei may involve different factors in bovine zygotes since they respond differently to the kinase modulations. Mol. Reprod. Dev. 52:43–49, 1999. © 1999 Wiley‐Liss, Inc.     

Hypo-osmotic swelling can accurately assess the viability of nonmotile sperm

Viable, healthy sperm are preferred for oocyte fertilization with intracytoplasmic sperm injection. Currently, motility is the most widely applied measure of sperm viability. However, with this criterion, viable but immotile sperm are overlooked as candidates for micromanipulation. Supravital stains identify viable sperm but may be toxic to the gamete or embryo. We tested the hypothesis that hypo-osmotic swelling, developed to assess sperm membrane integrity, can accurately determine sperm viability. Thawed sperm from 12 fertile donors were exposed to a hypo-osmotic solution and to two supravital stains. A total of 2,010 sperm were assessed for tail coiling after a 30-min exposure to hypo-osmotic solution. By supravital stains, 31% of thawed sperm were viable; 23% showed tail coiling. Among coiled-tail sperm, 100% were viable by supravital stain. As a measure of viability, tail coiling exhibited a sensitivity of 30%, specificity of 100%, and positive predictive value of 100% compared to supravital stains. With a 60-min hypo-osmotic incubation, the specificity (89%) and positive predictive value (78%) decreased significantly (P < 0.05). Therefore, hypo-osmotic swelling accurately detects viable sperm among a nonmotile population. Assay accuracy, however, is very sensitive to the incubation time in hypo-osmotic solution. Mol. Reprod. Dev. 47:200–203, 1997. © 1997 Wiley-Liss, Inc.     

Social and seasonal influences on reproductive biology in male moustached tamarins (Saguinus mystax)

In several primate taxa there is evidence that the social and physical environment can exert a significant effect on reproductive behavior and biology. In this paper we examine social and physiological factors influencing group composition and reproduction in free-ranging moustached tamarin monkeys (Saguinus mystax mystax). This species is characterized by cooperative care of the young and a breeding system that includes both polyandrous and polygyandrous matings. Body measurements collected on adult males residing in multimale groups indicate marked within-group differences in testes volume. In 12 of 17 groups examined, testes volume of at least two resident adult males differed by 21–174%. Among these males, testes volume was not correlated with either body weight or adult age class. We also examine whether factors such as time of year had an effect on reproductive condition. An analysis of body measurements of 128 adult male and 127 adult female moustached tamarins, wild-trapped and released in northeastern Peru, indicates cyclic changes in genital size. For males, mean monthly testes volume in July (712 mm³) was twice that recorded in June (351 mm³). Females exhibited a similar pattern. Although endocrine information on intra- and intersexual social effects on fertility are unavailable for S. mystax, given the high degree of social cooperation and lack of overt aggression among adult male group members, we offer the possibility that resident male moustached tamarins compete for access to the groups' lone breeding female through socially induced reproductive suppression and sperm competition. © 1996 Wiley-Liss, Inc.     

Effect on the immune system of mice exposed chronically to 50 Hz amplitude-modulated 2.45 GHz microwaves

The effect of continuous (CW; 2.45 GHz carrier frequency) or amplitude-modulated (AM; 50 Hz square wave) microwave radiation on the immune response was tested. CW exposures (6 days, 3 h/day) induced elevations of the number of antibody-producing cells in the spleen of male Balb/c mice (+37%). AM microwave exposure induced elevation of the spleen index (+15%) and antibody-producing cell number (+55%) in the spleen of male mice. No changes were observed in female mice. It is concluded that both types of exposure conditions induced moderate elevation of antibody production only in male mice. © 1996 Wiley-Liss, Inc.     

Can relaxed time constraints on sperm production eliminate protandry in an ectotherm?

In most temperate-zone reptiles, as in many other ectothermic taxa, males emerge from periods of inactivity (e.g. hibernation) before females, a pattern referred to as protandry. In the large body of theory depicting its evolution it is assumed that as selection pressures on females moderate female emergence time, net sexual selection on males shifts male emergence time accordingly. This is because early-emerging males might (i) benefit from advantages in behavioural interactions (e.g. obtain more matings, better territories, or a higher social status), or (ii) produce and mature more spermatozoa before the mating season. These putative advantages of early emergence occur simultaneously in most temperate-zone reptile species, because sperm production and copulations occur soon after emergence from winter inactivity. Thus, it is difficult to distinguish between the two hypotheses. To do so would require a system in which behavioural interactions occur at one time of year and sperm production at another. The southern snow skink ( Niveoscincus microlepidotum ) provides such an example; adult males fight with each other immediately after emerging from hibernation, but examination of the testes and epididymes of males throughout the year show that males do not produce spermatozoa until around 3 months later. Thus, the 'behavioural interactions' hypothesis predicts protandry in snow skinks, whereas the 'sperm production' hypothesis predicts synchronous spring emergence in males and females. Our field data, collected over 5 years, show that emergence dates in spring are the same for male and female skinks. Hence, when the selection pressure for rapid sperm production is relaxed, we no longer see the protandric emergence pattern characteristic of most temperate-zone reptiles.     

Characterization and genetic mapping of a mutation (ms35) which prevents anther dehiscence in Arabidopsis thaliana by affecting secondary wall thickening in the endothecium

The male sterile mutant, ms35 , of Arabidopsis thaliana was produced by X-irradiation of seeds. The mutant produces fertile pollen, but is male sterile because the anthers do not dehisce. Anther development in ms35 plants occurs as in wild-type Arabidopsis until shortly after microspores are released from meiotic tetrads. Thereafter, in the wild type, bands of lignified, cellulosic secondary wall thickenings are laid down around the cells of the anther endothecium. In contrast, wall thickenings are not formed in the endothecium of the ms35 mutant. Development of other lignified tissues, for example the vascular tissue of the stamen, occurs normally in ms35 plants. In mutant anthers, as pollen maturation is completed, the stomium is cleaved but the anther wall does not retract to release pollen. The block in anther dehiscence in ms35 plants is specifically correlated with the absence of endothecial wall thickenings. The ms35 mutation represents the first genetic evidence in support of the proposed role of the endothecium in anther dehiscence. The ms35 gene was mapped to the top arm of chromosome 3 ( hy2 -(4.17±2.31 cM)- ms35 -(32.14±5.45 cM)- gl1 ).     

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line. Received:15 December 1998 / Accepted:30 January 1999