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Androgen production by minced testis from adult rabbits was measured in response to a variety of FSHs and LHs from eutherian (sheep, rat, rabbit) and metatherian (kangaroo) mammals, a bird (turkey), reptiles (turtle, alligator) and an amphibian (frog). LHs from all three eutherian species were highly potent and produced clear stimulation at doses below 50 ng/ml; relative potencies in the rabbit were consistent with estimates from OAAD assay, but an anuran ovulation assay indicated considerable differences in potency among these three preparations. LH from the kangaroo had only about 4 percent the activity of the eutherian LHs in the rabbit testis. Potencies of mammalian (rat and ovine) FSHs were only a small fraction of the LHs (<1 percent); inactivation of ovine FSH by an LH-antiserum indicated that this low level of activity was due to residual LH contamination.Amphibian, reptilian and avian hormones were active in the rabbit testis, but a sharp dichotomy in potency was evident between these and all mammalian hormones. Nonmammalian hormones stimulated androgen production (with comparable ratios of testosterone and dihydrotestosterone), and maximal levels of androgen produced were the same or greater than obtained with mammalian preparations. However, nonmammalian hormones were consistently three to four orders of magnitude less potent than the eutherian LHs. Furthermore, in several tests, nonmammalian FSHs were also active in the minced rabbit testis at levels (20–110 percent of homologous LH) that could not be readily attributed to LH contamination. Comparisons with relative potencies for the same preparations in an anuran ovulation assay confirm that pronounced phylogenetic specificity underlies the differences in potencies between mammalian and nonmammalian gonadotropins.

The fibrous sheath is a unique cytoskeletal component in the principal-piece segment of the mammalian sperm flagellum. Monoclonal antibody ATC was shown by indirect immunofluorescence (IIF) to bind to the principal piece of the flagellum of permeabilized mouse, rat, and hamster sperm, but not to that region of guinea pig, rabbit, or human sperm. IIF on isolated fibrous sheaths confirmed that the antigen was present in the fibrous sheath of mouse, rat, and hamster sperm. On Western blots of mouse spermatozoa, ATC identified a relatively insoluble major antigen with an apparent molecular weight of 67,000 (Mr67,000). Hamster sperm fibrous sheaths contain an antigen of Mr66,000, while rat sperm fibrous sheaths contain an antigen of Mr65,500. The antigen was first detected in late spermatids, as determined by immunohistochemical procedures on sections of mouse, rat, and hamster testis. The antigen was not detected on Western blots of mouse brain, kidney, liver, or thymus. These results indicate that ATC recognizes a protein integral to the fibrous sheath of the principal piece of sperm detected by immunohistochemistry late in spermiogenesis that is probably restricted to the male germ cell line.

We have generated a monoclonal antibody directed against an antigenic determinant appearing on the surface of mouse sperm tails during passage through the epididymis (a determinant that we now term sperm maturation antigen number four [SMA 4]). The present study demonstrates that sperm retained in the ductuli efferentes following ligation do not acquire the antigen, suggesting that its appearance is not due to changes intrinsic to the sperm, but that the epididymal environment is required. To examine the role of the epididymis in the appearance of this antigen, sections of unfixed frozen or fixed, paraffin embedded tissue from different regions of the male reproductive tract have been studied by indirect immunofluorescence. Results indicate that the antigen is a secretory product of the epididymal epithelium, produced in a short segment of the distal caput epididymidis. Ligation experiments show that absence of sperm or testicular fluid from the epididymis does not affect production of this antigen. Examination of prepubertal mice indicates that antigen production is age dependent, production beginning in the epididymis in mice between 2 and 4 weeks of age. Indirect immunofluorescence analysis of sections of a variety of tissues and organs shows that the antigen is restricted to sperm and to epithelial cells of the male reproductive tract. Finally, experiments comparing the antibody-induced agglutinability of sperm from the caput epididymidis to that of sperm from the cauda epididymidis gives further evidence that the antigen resides on the sperm surface.