Murray R. Bakst

Peripheral androgen concentrations and testicular morphology in embryonic and young male Japanese quail

Abstract Plasma samples and gonads from male Japanese quail embryos were examined to determine the association of serum androgen concentrations and differentiation of steroidogenic cells. Embryos were sampled at 1- and 2-day intervals and seven posthatch samples were taken between 1 and 24 days of age. Plasma samples were analyzed by radioimmunoassay specific for testosterone. Gonadal samples were examined by electron microscopy. Plasma androgen was elevated on the eighth day of the 17-day incubation, declined, and rose again between 13 days of incubation through 3 days posthatch. Circulating androgen subsequently remained low until after 18 days of age. Interstitial cells in the embryonic quail testes retained a fibroblast-like appearance through hatching. Between 2 and 3 weeks posthatch interstitial cells differentiated into presumptive Leydig cells, as characterized by smooth endoplasmic reticulum and lipid droplets. Differentiation at that time appeared to be coincident with the increasing testosterone concentrations.

Structure of the Avian Oviduct With Emphasis on Sperm Storage in Poultry

The macroanatomy, histology, and fine structure of the avian oviduct is reviewed and related to its role in fertile egg production. The avian oviduct functions as a biological assembly line, beginning sequentially with the deposition of the albumen around the fertilized or unfertilized ovum, then the shell membrane, and lastly, the shell, all within 25 hr of ovulation. While in transit through the oviduct, the fertilized ovum progresses to the pre-gastrulation stage of development.

Restoration of Spermatogenesis and Male Fertility by Transplantation of Dispersed Testicular Cells in the Chicken

Abstract Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipients testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa. Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.

Apical blebs on sperm storage tubule epithelial cell microvilli: Their release and interaction with resident sperm in the turkey hen oviduct

Located at the anterior end of the turkey hens vagina are numerous discrete tubular invaginations of the surface epithelium, collectively referred to as the sperm storage tubules (SSTs). After mating or artificial insemination, sperm ascend the vagina, enter the SSTs, and over the ensuing days and weeks, gradually exit the SSTs and are transported to the anterior end of the oviduct to fertilize a daily succession of ova. Little is known regarding the cellular and molecular mechanisms responsible for sperm subsistence in the lumen of the SST. In this study, the origin of microvillus blebs (MvBs) on the apical tips of SST epithelial cells was examined, and their possible role in sperm survival was discussed. Regardless, if sperm are present or not, transmission electron microscopy revealed two types of microvilli differentiated by the presence or absence of pleomorphic unilaminar MvBs localized to their apical tips. Although some MvBs appeared to be discharging their contents into the SST lumen, others appeared to have pinched off the microvillus stem. When SSTs contained clusters of densely packed sperm, the sperm heads of those sperm adjacent to the SST epithelial cell surface were surrounded by the microvilli. Associated with the plasmalemma of sperm throughout the SST lumina were membrane fragments and small vesicles (30-130 nm in diameter), some of which appeared to have fused with sperm. It is concluded that the MvBs are a form of shedding vesicle released from the SST epithelial cell microvilli by apocrine secretion. On the basis of observations described herein and those of other authors, it is suggested that the MvBs contribute to sustained sperm storage in the SSTs by (1) supplying metabolic substrates used by resident sperm, (2) serving as fusogenic vehicles providing exogenous macromolecules that reversibly suppress sperm functions associated with fertilization (decapacitation?) and stabilize the sperm plasmalemma, and (3) acting as transport vesicles actively transporting fluid from the SST epithelial cells to the SST lumen.

Identification of various testicular cell populations in pubertal and adult cockerels

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.

Serum testosterone concentration during two breeding cycles of turkeys with low and high ejaculate volumes

Abstract Serum testosterone, ejaculate volume, sperm concentration, and total sperm per ejaculate were followed from 24 to 80 weeks of age in Large White breeder turkeys. Blood was collected immediately after the weekly semen collection and serum testosterone was determined by radioimmunoassay. Males were classified as low semen producers (LSP; ejaculate volume .50 ml). Semen production, which had started in all males at 28 weeks of age, stopped at 57 ± 2.4 and 62 ± 1.6 weeks, and resumed at 72 ± 0.2 and 71 ± 0.6 weeks (mean ± SEM) for LSP and HSP, respectively. Semen production (volume, concentration, and total sperm) of the second cycle was comparable to that of the first cycle. However, serum testosterone ranged from

Expression of the chicken GDNF family receptor α-1 as a marker of spermatogonial stem cells.

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipients testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.

EFFECTS OF ISOLATION AND CULTURE OF TURKEY PRIMARY FOLLICULAR OOCYTES ON MORPHOLOGY AND GERMINAL VESICLE INTEGRITY

A novel approach to the production of transgenic poultry is to use primary follicular oocytes (PFOs). However, fundamental information regarding the impact of isolation and culture procedures on PFO integrity is lacking. This study describes the isolation and culture of PFOs from mature turkeys and the effects of these procedures on PFO morphology and germinal vesicle (GV) integrity. To isolate PFOs, ovarian cortex was incubated in trypsin-EDTA alone or further incubated in collagenase plus hyaluronidase (CH). About 200 to 500 PFOs, ranging in size from less than 100 microns in diameter to 1,000 microns, were recovered from each ovary. The culture of PFOs less than 100 microns in diameter for 4 h resulted in blebbing of the oolemma followed by extrusion of ooplasm. Primary follicular oocytes 100 to 250 microns in diameter survived culture for 24 h whereas larger PFOs survived for up to 7 d. Those PFOs with intact granulosa cell investments survived longer than those fully or partially denuded of granulosa cells with CH. Co-culture of PFOs (100 to 250 microns in diameter) on a monolayer of granulosa cells derived from mature, yellow-yolk follicles augmented PFO survival rates. The rate of GV breakdown was not influenced by the isolation or culture of the PFO. These data provide the basis for developing procedures for the in vitro maturation and in vitro fertilization of isolated PFOs.

A comparison of Mott cell morphology of three avian species. II. - Bad behavior by plasmacytes?

&NA; Mott cells are atypical plasmacytes recognized microscopically by endoplasmic reticulum (ER) distensions (Russell bodies) a result of retained secretory product (antibody). Originally associated with parasitism, they are observed in a broad spectrum of immunopathology, sometimes involving hypergammaglobulinemia. Few descriptions of Mott cells appear in avian literature. The purpose of the manuscript is to provide examples identified by light microscopy in three poultry species. Transmission electron micrographs (TEM) of plasmacytes from the turkey oviduct mucosa are included for comparison with Mott cell light microscopic images. Wrights stained blood and bone marrow from commercial and specific pathogen free (SPF) chickens, ducks, and turkeys are the sources. Mott cell positive samples commonly occurred with leukocytosis or leukemoid reactions, polymicrobial bacteremia, and fungemia. Atypical granulocytes and leukocytes regularly accompanied Mott cells. It is proposed that circulating Mott cells are “sentinels” indicative of stress, dyscrasia, and pathology. Moreover, Mott cells, like other atypia, complicate the interpretation of simple heterophil/lymphocyte (H/L) ratios. As Mott cells are defective plasmacytes these observations address hematology, immunology, pathology, and welfare issues.