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Müllerian Inhibiting Substance (MIS) causes regression of the Müllerian ducts during a critical period in embryonic development in male mammals. In Persistent Müllerian Duct Syndrome (PMDS), an autosomal recessive trait in humans and dogs, the Müllerian ducts fail to regress in otherwise normal males. Previously we reported that PMDS-affected dogs produce bioactive testicular MIS postnatally. The purpose of the present study was to determine whether PMDS-affected canine embryos appropriately express MIS mRNA and protein during the critical period for Müllerian duct regression. Homozygous (PMDS-affected) and normal canine embryos were removed from timed pregnancies. Gonadal sex and the degree of Müllerian duct regression were determined from histologic sections. Positive immunohistochemical staining for MIS was found in testis sections of PMDS-affected and normal male embryos. A 1.8-kb MIS mRNA transcript was detected in testes of PMDS-affected males and normal male embryos and neonates. Furthermore, equal amounts of MIS mRNA transcript were detected in testes of PMDS-affected embryos and normal male littermates during the critical period for Müllerian duct regression. These data support a hypothesis of target organ resistance, such as an abnormality in the putative MIS receptor, as the etiology of the defect in this dog model.

Polypeptides secreted by uterine tube epithelial cells (UTEC) may facilitate sperm cell capacitation in vivo. This experiment evaluated the effect of sperm-UTEC co-culture on de novo protein synthesis by epithelial cells of the tubal isthmus. Comparisons of the patterns of proteins secreted into medium were made between four culture groups incubated for 24 h in the presence of 35S-methionine: group 1, sperm cells alone; group 2, control UTEC monolayers; group 3, UTEC co-cultured with sperm cells; and group 4, UTEC partitioned by a diffusible membrane from sperm cells during culture. Two-dimensional PAGE followed by fluorography was used to analyze conditioned medium containing secreted proteins from each group. The experiment was replicated four times. Sperm cells alone secreted no detectable proteins, whereas control UTEC monolayers produced a wide array of polypeptides. Sperm cells attached to UTEC in co-culture within minutes, and the resultant protein profile for these UTEC differed markedly from that of the control UTEC. Several new proteins were seen only from co-cultured cells, whereas other protein groups that were present with UTEC alone were absent in the co-culture medium of group 3. The protein pattern expressed by UTEC partitioned from sperm cells (group 4) was intermediate between that of the group 2 controls and that of co-cultured UTEC (group 3). In summary, the attachment of sperm cells to the UTEC during co-culture changed the types and quantities of proteins secreted into the conditioned medium as compared to those of control UTEC monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

The embryonic period during which Mullerian duct regression and Mullerian Inhibiting Substance (MIS) secretion occur was determined in canine embryos removed from timed pregnancies (32, 36, 37, 39, 42, and 46 days gestation). Sex chromosomes of each embryo were identified in metaphase spreads prepared from fibroblast cultures. Testicular differentiation, defined by seminiferous tubule formation and the presence of Sertoli cells and Leydig cells, and the degree of Mullerian duct regression were determined by careful morphologic analysis of histologic sections of canine embryonic gonads (n = 20) and Mullerian ducts (n = 20). MIS was detected immunohistochemically in embryonic testes using avidin-biotin complex enhancement of a specific rabbit polyclonal anti-MIS antibody. Testicular differentiation was observed at 36 days gestation. The earliest evidence of Mullerian duct regression in male embryos was observed at 36 days gestation, and regression was completed by 46 days gestation. Positive staining for MIS was present in testes from 36 to 46 days (n = 9). Staining was absent in the undifferentiated testis (n = 1) at 32 days gestation and in ovaries at all ages tested (n = 10). Thus, MIS is normally present throughout the critical period for Mullerian duct regression in the embryonic male dog.

Breeding studies in a strain of miniature schnauzer dogs with Persistent Müllerian Duct Syndrome (PMDS) indicate this syndrome is inherited as an autosomal recessive trait, as it is in man. Testes of neonatal dogs affected with PMDS and normal male littermates were examined for Müllerian Inhibiting Substance (MIS) production by immunohistochemistry and bioassay. MIS immunoactivity was detected in Sertoli cells of normal and affected pups using an avidin-biotin complex-enhanced method. Rat embryonic Müllerian ducts regressed when cocultured with testis fragments of both normal and affected pups in a graded organ culture bioassay, demonstrating that the MIS produced was bioactive. These findings indicate that Müllerian duct persistence in affected dogs is not due to a mutation in the structural gene for MIS, but rather, by inference, to a failure of response to MIS at the receptor level.

In normal males, Müllerian Inhibiting Substance (MIS), produced by testes during an embryonic critical period, is thought to induce regression of the Müllerian duct system, including the oviducts and uterus. In XX sex-reversed dogs, an apparent contradiction has been reported: The uterus persists in the presence of testes or ovotestes. The objective of this study is to determine whether testes of XX male and ovotestes of true hermaphrodite dogs produce MIS, and to examine the anatomy of Müllerian duct derivatives of affected dogs for evidence of regression. Gonadal samples were tested for MIS activity in a bioassay. The mean MIS activity score of XX males was similar to that of normal XY males and significantly greater than that of normal XX females. The mean MIS activity score of XX true hermaphrodites was intermediate between normal XX females and XY males. Within the true hermaphrodite group, ovotestes in which the proportion of testicular tissue was greater than or equal to 1/2 had higher MIS scores than those in which the proportion of testicular tissue was less than 1/2. XX males had a well-developed epididymis adjacent to each testis, but no oviducts. In true hermaphrodites, the oviduct regressed and an epididymis was present when greater than or equal to 1/2 of the adjacent ovotestis was testicular, and MIS activity in that gonad was high. A few ovotestes with intermediate levels of MIS activity had both an oviduct and an epididymis. Regression of the oviductal portion of the Müllerian duct system was positively correlated to the amount of testicular tissue and the MIS activity of the gonad, as would be predicted by Jost's original hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)