Cell membrane and mitotic markers show that the neonatal rat gubernaculum grows in a similar way to an embryonic limb bud

How the gubernaculum guides the testis into the scrotum remains controversial, with various proposals from passive inversion to active growth. We aimed to determine if the gubernaculum contains an area of active proliferation, such as a "progress zone" in a growing embryonic limb bud, using a fluorescent cell membrane marker, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC12(3)], to trace cell migration, and 5-bromodeoxyuridine (BUDR) (a thymidine analogue) as a mitotic marker.Gubernacula were collected from neonatal male rats (n = 42, day 1-2, Sprague-Dawley) and cultured with calcitonin gene-related peptide (CGRP; 714 nmol/L). 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-coated glass beads (diameter, 150-212 microm) were placed next to the bulb for the first 3 hours. Gubernacula were cultured for 3, 18, and 24 hours, then frozen sections cut and examined by confocal microscopy (wavelength, 549 nm). In a second experiment, pups not exposed to exogenous CGRP (n = 53, day 0, Sprague-Dawley) were injected intraperitoneally with BUDR (50 mg/kg of body weight); gubernacula were collected at 2, 48, 72, and 96 hours postinjection (PI), sectioned, and stained using immunohistochemistry to count the number of BUDR-positive cells per 100 cells (labeling index) in the bulb, cremaster, cord, and epididymis.After 24 hours' culture with CGRP, the bulb showed an oval region (diameter, 300 microm) of high fluorescence, and the cremaster region showed elongated cells migrating out of the bulb. When cultured without CGRP, the same oval region contained no fluorescence. In vivo BUDR labeling index increased in all areas until 48 hours postinjection and then decreased most rapidly in the bulb (P.05), in the presence of endogenous CGRP from the genitofemoral nerve.The rat gubernaculum contains a putative progress zone, such as in a growing limb bud, in the presence of CGRP. Cells migrate out of this zone to form cremaster muscle. We hypothesize that proliferation in the bulb elongates the gubernaculum, whereas proliferation of cremaster cells would increase gubernacular diameter. This brings to "life" the gubernaculum as an actively growing organ in contrast to the inert ligament connecting the testis to the scrotum portrayed in most anatomy textbooks.