The photoaffinity analog [32P]8-N3 cAMP (8-azido adenosine 3',5'-monophosphate was used to analyze the membrane sidedness of rat sperm cAMP binding proteins during epididymal maturation. Evidence is presented here which supports the hypothesis that 35-45% of the regulatory subunits of the Type I and Type II cAMP-dependent protein kinases are readily available to externally added cyclic nucleotide. It was observed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and autoradiography that only two rat sperm proteins (Mr = 49K and 55K) were photolabeled which comigrated on gels with partially purified Type I and Type II regulatory subunits, respectively. Both of these photolabeled epididymal sperm proteins were saturated at physiological titers of [32P]8-N3cAMP and photoincorporation of [32P]8-N3 cAMP was specific since other SDS-resolvable sperm proteins did not photoincorporate the analog. Caput and cauda sperm protein photoincorporation could be effectively blocked by low levels of cAMP, but not by cGMP, ATP or GTP. Sperm epididymal maturation coincided with changes in the cAMP-dependent protein kinase subunits since cauda sperm contained more available Type II than did caput sperm. A subcellular analysis of cAMP-dependent protein kinase regulatory subunit in head and tail fractions was done for caput and cauda sperm and demonstrated that the tail fractions showed more photo-labeling of both Type I and II regulatory subunits than did the head fractions.

In the present investigation, comparative baseline information on selected sperm characteristics of ejaculate spermatozoa of the domestic (Mustela putorius furo), fitch (Mustela sp.) and black-footed ferrets (Mustela nigripes) and the Siberian polecat (Mustela eversmanni) are presented. The main emphasis was to establish differences and similarities among these species in relation to semen and sperm quality during the breeding season, in cryopreservation success and in supporting sperm motility in different extenders or physiological media. The results confirm that most sperm morphology abnormalities were evident during the beginning of the breeding cycle in all four species. No significant interspecies differences were apparent in the sperm attributes examined, for all sampling months during the breeding season. Moreover, all species exhibited comparable patterns of reproductive seasonality. Cryopreservation suppressed sperm characteristics equally in all species studied. Ejaculate spermatozoa of closely related ferret species shared many similar motion characteristics using computer-aided sperm motility analysis. These results suggest that the basic sperm physiology of the ferret species under examination is very similar. Disparate to the interspecies comparisons, there were significant differences for most sperm motion parameters when spermatozoa of any of the ferrets were compared in different extenders. Assisted reproductive technologies developed for use in domestic ferret, fitch ferret or Siberian polecat may be successfully applied to captive breeding of the black-footed ferret using semen during any of the functional breeding months.

The photoaffinity probe (32P) 8-N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP-dependent protein kinase (cAMP-PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell-free seminal plasma was found to be free of detectable (32P) 8-N3 cAMP-binding proteins. The 8-N, cAMP was also effective in stimulating endogenous cAMP-PK activity in intact and disrupted sperm. A substantial amount of (32P) 8-N3 cAMP binding to types I and II regulatory subunits and cAMP-PK activity was detected on washed intact cells. Intact cells bound 1.80 pmol of (32P) 8-N3 cAMP/mg protein and had cAMP-PK activity of 824 units/10(8) cells. Disrupted cells bound 3.95 pmol (32P) 8-N3 cAMP/mg protein and had a cAMP-PK activity of 2,206 units/10(8) cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8-N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.

The development of the seminal vesicle from the mesonephric duct is described. Particular attention is given to the recent biochemistry of seminal vesicle proteins. Proteins in the seminal vesicle fluid are few in number, may be insoluble at certain pH, and frequently form large macromolecular aggregates. Although not an absolute requirement for fertility, seminal vesicle fluid assists in a number of ways to insure fertility. A biochemical model is presented that demonstrates that CAMP dependent phosphorylation may be an important interaction between sperm and certain seminal vesicle proteins.Key Words: development, secretory proteins, seminal vesicle, sperm phosphorylation. INTRODUCTIONSeminal vesicles are secretory organs of the male genital tract found in certain eutherian mammals. The size, chemical composition of the secretions, and morphological characteristics of the seminal vesicles are variable, species ~p e c i f i c , '~.~* and dependent on androgens for embryonic development, growth, and secretory f~n c t i o n .~~.~~ EMBRYONIC DEVELOPMENTDuring embryonic development the seminal vesicles (and the epididymis and ductus deferens) are derived from the mesonephric or WolffianIn mice (on the 15th day of gestation) and rats (on the 16th day of gestation), there is a notable expansion of the epithelium of the lower aspect of the Wolffian duct. This marks the site where the seminal vesicle rudiment will bud off in later g e~t a t i o n . '~ Development of this tissue is dependent on testosterone secretion by the fetal testis. Testosterone prevents programmed cell death and stimulates growth and organogenesis of the mesonephric duct deri~ates.~' The onset of testosterone formation by the fetal testis occurs before male differentiation of the urogenital tract.8 The probable source of the steroid substrates (pregnenolone and progesterone) used in testosterone synthesis in the testis is the placenta or the adrenal gland.35Wolffian duct stabilization and morphogenesis of the seminal vesicle results from an androgen-dependent interaction between embryonic mesenchyme and epithelial tissues. l 6 Dur- In the neonatal rodent there are two peaks of seminal vesicle cell proliferation, from birth to approximately fifteen days of age, and just before puberty (twenty-five-thirty-five days). In the intervening time period there is a quiescent interval.25 The response of the seminal vesicles to androgen at puberty is enhanced by the neonatal exposure to androgens, an irreversible phenomenon known as imprinting.42 The biphasic response of the seminal vesicles seems to be the result of two populations of Leydig cells during development. One population appears during fetal life (secretes fetal testosterone) and the other arises during puberty.26 At birth the fetal Leydig cells undergo regression. The regression involves reversion of these cells to a less differentiated cell type rather than cell death. The tissue then enters the quiescent period.During the period of regression the testis secretes 5 alpha androgens (androsterone...

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