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R. P. AMANN

Summary.

Six Dutch-type and nine New Zealand White rabbits were each ejaculated at frequencies of 1 × 12 hr, 1 × 24 hr and 2 × 48 hr. Ejaculate volume and sperm concentration were lower (P<0·01) at 1 × 12 hr than at either of the other two frequencies. Daily sperm output did not differ significantly among the three frequencies. Dutch-type rabbits had a significantly lower daily sperm output than New Zealand Whites.

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R P Amann and D N R Veeramachaneni

Cryptorchidism is failure of one or both testes to descend into the scrotum. Primary fault lies in the testis. We provide a unifying cross-species interpretation of testis descent and urge the use of precise terminology. After differentiation, a testis is relocated to the scrotum in three sequential phases: abdominal translocation, holding a testis near the internal inguinal ring as the abdominal cavity expands away, along with slight downward migration; transinguinal migration, moving a cauda epididymidis and testis through the abdominal wall; and inguinoscrotal migration, moving a s.c. cauda epididymidis and testis to the bottom of the scrotum. The gubernaculum enlarges under stimulation of insulin-like peptide 3, to anchor the testis in place during gradual abdominal translocation. Concurrently, testosterone masculinizes the genitofemoral nerve. Cylindrical downward growth of the peritoneal lining into the gubernaculum forms the vaginal process, cremaster muscle(s) develop within the gubernaculum, and the cranial suspensory ligament regresses (testosterone not obligatory for latter). Transinguinal migration of a testis is rapid, apparently mediated by intra-abdominal pressure. Testosterone is not obligatory for correct inguinoscrotal migration of testes. However, normally testosterone stimulates growth of the vaginal process, secretion of calcitonin gene-related peptide by the genitofemoral nerve to provide directional guidance to the gubernaculum, and then regression of the gubernaculum and constriction of the inguinal canal. Cryptorchidism is more common in companion animals, pigs, or humans (2–12%) than in cattle or sheep (≤1%). Laboratory animals rarely are cryptorchid. In respect to non-scrotal locations, abdominal testes predominate in cats, dogs, and horses. Inguinal testes predominate in rabbits, are common in horses, and occasionally are found in cats and dogs. S.c. testes are found in cattle, cats and dogs, but are most common in humans.

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L. J. LINNETZ and R. P. AMANN

Summary.

Fifteen rabbits were allotted equally to three groups: (I) two successive ejaculations every 48 hr, (II) sexual rest, and (III) unilateral vasectomy followed by sexual rest. One month later they were killed. Acid phosphatase was found in the Golgi area of epididymal columnar cells. Scattered columnar cells with intense acid phosphatase activity were also found in epididymal regions 1 and 6 and they were most numerous after vasectomy. Alkaline phosphatase was found apically in epididymal columnar cells and in their stereocilia. The weaker reaction in the proximal ductus deferens and epididymal regions 7 and 8 of the ejaculated rabbits supports the concept that regular ejaculation diminishes the rate of sperm resorption within the epididymis. The ampullae of ejaculated rabbits contained more alkaline phosphatase than those of non-ejaculated animals. Cytoplasmic pas-positive droplets were most abundant in regions 1 and 7 of the epididymis. Glycogen was found only in region 6. Little evidence was found for a holocrine cell cycle in the epididymis. Large, pas-positive cells, considered to be phagocytes, were most common in regions 1 and 6 of the epididymis and were present in increased numbers after vasectomy. Phagocytes may resorb materials emanating from the testis.

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L. D. S. BARKER and R. P. AMANN

Summary.

Sites of protein absorption and secretion in the epididymis and changes in sperm antigenicity during their maturation and senescence were investigated. Tissue and sperm samples from normal bulls, unilaterally vasoligated bulls and bulls in which one epididymis was ligated distal to the caput epididymidis were examined using fluoresceinconjugated immune globulins. The globulins were obtained from antisera against spermatozoa from bull cauda epididymidis, ejaculated spermatozoa, plasma from the cauda epididymidis and seminal plasma. In mature spermatids, acrosomal immunofluorescence was homogeneous. After spermatozoa had traversed the first half of the caput epididymidis, the apical body was especially prominent. Specific immunofluorescence of the post-nuclear cap, cytoplasmic droplet, midpiece, and principal piece was not observed except in presumably senescent spermatozoa from spermatocoeles.

Non-ciliated, antigen-secreting cells were detected by their immunofluorescence in the epithelium of the efferent ducts within the proximal caput epididymidis. These secretory cells produce sperm-coating proteins which are antigenically similar to certain components of seminal vesicle fluid. Immunofluorescence of the apical cytoplasm and stereocilia in the proximal caput epididymidis indicated that this epithelium probably absorbed sperm-specific proteins emanating from the testis.

Immunofluorescence in the apical cytoplasm of the epithelium lining the corpus and proximal cauda epididymidis was suggestive of secretory activity. Similar fluorescence in the epithelium of the vas deferens may have resulted from the absorption of proteins.

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R. P. AMANN and J. T. LAMBIASE Jr

Although male rabbits are frequently used for research on reproduction, certain questions must be answered to facilitate the design of meaningful experiments. Two of these questions are discussed in the present paper, namely: when is a male sexually mature in terms of sperm production or sperm output?, and what are norms for semen characteristics and daily sperm output (dso) ?

Twelve random-bred New Zealand White bucks, born between 26th June and 2nd July 1965, were purchased at 17 weeks of age. They were exposed to natural daylight and ambient temperature between 0 and 30° C. During postnatal Weeks 18 and 19, rabbits were trained to ejaculate with a Waltontype artificial vagina. Live teaser does were used. From

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G. J. KILLIAN and R. P. AMANN

Summary.

Hydroxysteroid dehydrogenases (HSD) were evaluated histochemically in tissues representing seven regions of the epididymides from twenty-five neopubertal and three adult bulls. The epithelium of the epididymides was devoid of HSD activity against each of twenty steroid substrates tested; only 3β,16β-dihydroxy-5-androsten 3-methyl ester was dehydrogenated. In luminal spermatozoa, however, 3α-, 3β-, 16β- and 17β-HSD activities were detected in sections from most neopubertal and all adult bull epididymides incubated with 5β-androsterone, 5β-epiandrosterone, 3β,16β-dihydroxy-5-androsten 3-methyl ester and 5β-dihydrotestosterone. All other substrate incubations were negative. There was no age-associated difference in demonstrable HSD activity of epididymal spermatozoa. In smears of unwashed testicular spermatozoa from adult bulls, 3α-HSD activity was most conspicuous on the sperm head and cytoplasmic droplet, 3β-HSD activity was most prevalent in the mid-piece and the 17β-HSD activity predominated in the mid-piece and the cytoplasmic droplet.

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L. D. S. BARKER and R. P. AMANN

Summary.

Immunodiffusion and immunoelectrophoretic analyses, including absorption tests, were used to determine the heterogeneity of antigens in bull spermatozoa and reproductive fluids and to characterize antisera against these antigens. Antisera were produced against bull seminal plasma, seminal vesicle fluid, cauda epididymal plasma, blood serum, washed ejaculated spermatozoa, washed cauda epididymal spermatozoa and sperm fractions. Four sperm-specific antigens were detected. One antigen was located in the sperm head, one in the sperm tail and two others were common to both the head and tail. The tailspecific antigen was detected only after mechanical rupture of washed spermatozoa. The major antigens of spermatozoa are localized in the cell membrane and acrosome. Apparently, sperm antigens were present in cauda epididymal plasma and seminal plasma. These antigens may have been released by physiologically normal spermatozoa or they may represent end products of sperm dissolution within the epididymis and vas deferens. Their presence makes immunological analyses of reproductive fluids difficult.

Cauda epididymal plasma and seminal vesicle fluid shared at least two antigens not present in blood serum. These antigens in cauda epididymal plasma coated the sperm cell and probably were secreted by the caput epididymidis. Coating antigens were more tightly bound to ejaculated spermatozoa than to spermatozoa from the cauda epididymidis.

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R. P. AMANN and J. O. ALMQUIST

Summary.

Gonadal and extra-gonadal sperm reserves were determined for nine 36-month-old Holstein bulls 23 weeks after unilateral vasectomy. The postoperative treatment groups, of three bulls each, were subjected to the following regimen: sexual rest, two ejaculates per week, and eight ejaculates per week for the last 20 weeks before slaughter. The correlation between gonadal reserves and testis weight was highly significant, +0·70. Gonadal reserves averaged 15·2 ± 0·8 × 109 and were not affected by either unilateral vasectomy or pre-slaughter ejaculation frequency. Sperm reserves in the caput and corpus epididymis also were unaffected by treatments. Sperm reserves in the cauda epididymis on the intact side were similar in the sexuallyrested and twice-weekly-ejaculated bulls. In bulls ejaculated eight times weekly, the cauda on the intact side contained 24% fewer spermatozoa just prior to ejaculation than if the bulls had been sexually rested. In eight of the nine bulls, sperm reserves in the cauda epididymis on the vasectomized side averaged 22·0 × 109 spermatozoa as compared to 11·4 × 109 in the contralateral cauda; an average of 17·8 × 109 spermatozoa accumulated in the ductus deferens just proximal to the site of vasectomy. In the other bull, the cauda epididymis on the vasectomized side contained 77·4 × 109 spermatozoa but there was no accumulation in the ductus deferens. After 23 weeks, some spermatozoa were found distal to the site of ligation in all nine bulls.

There appears to be a reciprocal relationship between sperm resorption and the total number of spermatozoa removed by collection and masturbation. The apparent rate of sperm resorption in the epididymis on the intact side of three bulls collected eight times weekly was 57%. In the vasectomized sides of nine bulls, it was greater than 96%. The rate of sperm resorption was postulated to be dependent upon the number of spermatozoa present in the cauda epididymis.

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B. L. GLEDHILL and R. P. AMANN

Earlier studies evaluating the condensation of chromatin during spermiogenesis revealed that deoxyribonucleoprotein (DNP) becomes more resistant to thermal denaturation as the round spermatid elongates and assumes the shape of a mature spermatozoon (Ringertz, Gledhill & Darżynkiewicz, 1970). Unfortunately, only heterogeneously aged populations of cells, represented by spermatids in loosely defined categories and at differing intervals before release from the germinal epithelium, could be studied. Homogeneous preparations of spermatozoa leaving the testis were unavailable. Because the `testicular spermatozoa' group evaluated by Ringertz et al. (1970) was a mixture of spermatids and spermatozoa, it was not possible to determine if final stabilization of germ-cell DNP occurs during the latter phases of spermiogenesis or in the caput epididymidis. From the data for two bulls, Gledhill (1972) suggested that the thermal denaturation profile for spermatozoa removed from the caput epididymidis was different from that for cells obtained from the ductus deferens. He suggested, therefore,

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F. R. Tekpetey, D. N. R. Veeramachaneni and R. P. Amann

Summary. Androgen receptor was immunolocalized in the epididymal epithelium of rams and in isolated cells using an antibody against a synthetic polypeptide representing a portion of the androgen receptor. Immunostaining was predominant in the epithelium in tissue sections. Concentrations of androgen receptor were determined in cells from the central caput, distal caput, and central corpus epididymidis enzymically dissociated and elutriated to provide two fractions. On the average (n = 18), Fraction I contained 8% principal cells while Fraction II contained 71% principal cells; the stromal cells in each fraction were primarily smooth muscle and fibroblasts. For each sample, the number of DHT receptors (fmol) per 106 total cells was greater in Fraction II than in Fraction I. Few cells in Fraction I were immunostained for androgen receptor, whereas most cells in Fraction II were intensely stained. The numbers of DHT receptors per cell, or per principal cell, were similar for the central caput and distal caput, but lower in the central corpus epididymidis. The results support our hypothesis that most epididymal DHT receptors are localized in principal cells and confirm that the region between the central caput and proximal corpus of the ram epididymis is most dependent on androgen stimulation.

Keywords: epididymis; androgen receptor; principal cells; ram