Generation of a familial hypercholesterolemia model in non-human primate.

Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.

Impact of administering umbilical cord-derived mesenchymal stem cells to cynomolgus monkeys with endometriosis.

Purpose: This study aimed to explore whether umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be used as a therapeutic resource for endometriosis. Methods: Of seven cynomolgus monkeys with endometriosis, five were administered UC-MSCs (intervention group) and two were administered saline (control group). First, intravenous US-MSC treatment was administered for three months. Second, weekly intravenous US-MSC administration combined with monthly intraperitoneal US-MSC administration was conducted for 3 months. Finally, weekly intraperitoneal US-MSC administration was conducted for 3 months. The dose of UC-MSCs was set to 2 × 106 cells/kg for all administration routes. Laparoscopic findings and serum cancer antigen 125 (CA125) levels were also evaluated. The Revised American Society for Reproductive Medicine classification was used for laparoscopic evaluation. Results: Laparoscopic findings showed exacerbation of endometriosis after intraperitoneal UC-MSC administration, although no changes were observed in the control group. Intravenous UC-MSC administration decreased the level of CA125 in all monkeys; however, the difference was not significant. Intraperitoneal UC-MSC administration significantly exacerbated endometriosis compared with intravenous administration (p = 0.02). Conclusions: This study revealed that intraperitoneal UC-MSC administration exacerbates endometriosis in a nonhuman primate model of the disease.

A new primate model of hypophyseal dysfunction.

For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey's general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

First Successful Delivery after Uterus Transplantation in MHC-Defined Cynomolgus Macaques.

Delivery following uterus transplantation (UTx)-an approach for treating uterine factor infertility-has not been reported in nonhuman primate models. Here, six female major histocompatibility complex (MHC)-defined cynomolgus macaques that underwent allogeneic UTx were evaluated. Antithymocyte globulin and rituximab were administered to induce immunosuppression and a triple maintenance regimen was used. Menstruation resumed in all animals with long-term survival, except one, which was euthanized due to infusion associated adverse reaction to antithymocyte globulin. Donor-specific antibodies (DSA) were detected in cases 2, 4, and 5, while humoral rejection occurred in cases 4 and 5. Post-transplant lymphoproliferative disorder (PTLD) developed in cases 2 and 3. Pregnancy was attempted in cases 1, 2, and 3 but was achieved only in case 2, which had haploidentical donor and recipient MHCs. Pregnancy was achieved in case 2 after recovery from graft rejection coincident with DSA and PTLD. A cesarean section was performed at full-term. This is the first report of a successful livebirth following allogeneic UTx in nonhuman primates, although the delivery was achieved via UTx between a pair carrying haploidentical MHCs. Experimental data from nonhuman primates may provide important scientific knowledge needed to resolve unsolved clinical issues in UTx.

The Natural History of Spontaneously Occurred Endometriosis in Cynomolgus Monkeys by Monthly Follow-Up Laparoscopy for Two Years.

Endometriosis, a disease in which endometrial tissue proliferates outside the uterus, is a progressive disease that affects women in reproductive age. It causes abdominal pain and infertility that severely affects the quality of life in young women. The mechanism of the onset and development of endometriosis has not been fully elucidated because of the complex mechanism involved in the disease. Nonhuman primates have been used to study the pathogenesis of spontaneous endometriosis because of their gynecological and anatomical similarities to humans. To reveal the natural history of endometriosis in cynomolgus monkeys, we selected 11 female cynomolgus monkeys with spontaneous endometriosis and performed monthly laparoscopies, mapping endometriotic lesions and adhesions up to two years. At the initial laparoscopy, endometriotic lesions were exclusively found in the vesicouterine pouch in 45.4% (5/11) of the monkeys and spread to the Douglas' pouch over time. Appearance of small de novo lesions and disappearance of some of the small lesions were observed in 100% (11/11) and 18.2% (2/11) of the monkeys, respectively. Endometriosis developed in all monkeys, and the speed of progression varied greatly among individuals that could be attributed to the degree or frequency of retrograde menstruation and genetic factors; these findings support the similarities between humans and monkeys, thus verifying the value of this nonhuman primate model. Finding reliable quantification markers and unravelling the underlying factors in correlation with the spatiotemporal development of the disease using a nonhuman primate model would be useful for the better management of endometriosis in humans.

Establishment of macaque trophoblast stem cell lines derived from cynomolgus monkey blastocysts.

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.

Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments.

Long-Term Outcome and Rejection After Allogeneic Uterus Transplantation in Cynomolgus Macaques.

Uterus transplantation (UTx) is an option for women with uterine factor infertility to have a child, but is still in the experimental stage. Therefore, allogeneic animal models of UTx are required for resolution of clinical issues. In this study, long-term outcomes were evaluated in four recipients (cases 1-4) after allogeneic UTx in cynomolgus macaques. Immunosuppression with antithymocyte globulin induction and a triple maintenance regimen was used. Postoperative ultrasonography and biopsy of the transplanted uterus and immunoserological examinations were performed. All four recipients survived for >3 months after surgery, but continuous menstruation did not resume, although temporary menstruation occurred (cases 1 and 2). All animals were euthanized due to irreversible rejection and no uterine blood flow (cases 1, 2 and 4) and post-transplant lymphoproliferative disorder (case 3). Donor-specific antibodies against MHC class I and II were detected in cases 1, 2 and 4, but not in case 3. Peripheral lymphocyte counts tended to elevate for CD3+, CD20+ and NK cells in conjunction with uterine rejection, and all animals had elevated stimulation indexes of mixed lymphocyte reaction after surgery. Establishment of allogeneic UTx in cynomolgus macaque requires further exploration of immunosuppression, but the clinicopathological features of uterine rejection are useful for development of human UTx.

Paromomycin sulfate is an effective treatment for balantidiasis in captive cynomolgus monkeys.

There are few effective antimicrobial agents against Balantidium coli infection. The effect of paromomycin sulfate (PS) against B. coli was confirmed in this study of 596 captive cynomolgus monkeys. In several trials, the minimum dose and duration of oral administration of PS were 25 mg/day for 5 + 5 days, with a 2-day withdrawal interval. To facilitate daily PS administration, pumpkin cakes supplemented with PS were made, which not only resulted in precise effects but also increased the efficiency of preparation and administration of PS by the animal care staff. No cysts or trophozoites were detected at 14 or 16 days after the last treatments. There were no obvious differences in blood and biochemical parameters between before and after administration of PS. These results indicate that PS is effective for elimination of B. coli without hematological side effects. These data could contribute to the control of microbiological pathogens during veterinary care and colony management in primate facilities.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

Poor second ovarian stimulation in cynomolgus monkeys (Macaca fascicularis) is associated with the production of antibodies against human follicle-stimulating hormone.

Cynomolgus monkeys (Macaca fascicularis) are a valuable model organism for human disease modeling because human physiology and pathology are closer to those of cynomolgus monkeys than rodents. It has been widely reported that mature oocytes can be recovered from cynomolgus monkeys through ovarian stimulation by human follicle-stimulating hormone (hFSH). However, it is unknown whether mature oocytes can be effectively obtained through a second ovarian stimulation by hFSH. Here, we report that some ovaries (eight ovaries from 14 female monkeys) were stimulated effectively by hFSH even after the first ovum pick up, whereas the others were stimulated poorly by hFSH. Furthermore, we found antibodies against hFSH only in the serum of female monkeys with poorly stimulated ovaries. Collectively, these data suggest that anti-hFSH antibodies in serum may cause a poor ovarian response to hFSH stimulation. Finally, detection of such antibodies as well as observation of the ovary over the course of hFSH administration might be useful to predict favorable second ovarian stimulation by hFSH.

Evaluation of allowable time and histopathological changes in warm ischemia of the uterus in cynomolgus monkey as a model for uterus transplantation.

INTRODUCTION: The objective of this study was to examine the allowable warm ischemic time and pathological changes due to ischemia and reperfusion injury in the uterus of the cynomolgus monkey as a model for uterus transplantation. MATERIAL AND METHODS: Six female cynomolgus monkeys were used in the study. The uterus was resected from the vaginal canal and connected through the bilateral ovarian and uterine arteries and veins only. One animal was used as a control. In the other five animals, the bilateral uterine and ovarian vessels were clamped for 0.5, 1, 2, 4 and 8 h, respectively. Biopsy of the smooth muscle tissue of corpus uteri was performed after each ischemic time and after subsequent reperfusion for 3 h. Biopsy samples were observed by light and electron microscopy. Menstruation recovery was monitored. RESULTS: There were no particular findings in both light and electron microscopy after ischemia for up to 2 h and after subsequent reperfusion. There were no marked changes after ischemia for 4 h, but dilated nuclear pores and rough endoplasmic reticulum swelling were found after reperfusion. These changes also occurred, along with mitochondrial swelling and cristae loss after ischemia for 8 h, and plasma membrane loss, nuclear fragmentation and chromatin condensation were found after reperfusion. Periodical menstruation restarted in all animals with ischemia up to 4 h, but the animal with ischemia for 8 h had amenorrhea and uterine atrophy. CONCLUSIONS: The uterus of the cynomolgus monkey tolerates warm ischemia for up to 4 h.

Protective efficacy of passive immunization with monoclonal antibodies in animal models of H5N1 highly pathogenic avian influenza virus infection.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

Efficacy of repeated intravenous administration of peramivir against highly pathogenic avian influenza A (H5N1) virus in cynomolgus macaques.

Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.

Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine.

PURPOSE: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. METHODS: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. RESULTS: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na-K adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for < or =2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. CONCLUSIONS: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea < or =2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.

Cultivated corneal endothelial cell sheet transplantation in a primate model.

PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (Macaca fascicularis).

In nonhuman primates (NHPs), there have so far been few reports about nuclear transfer (NT), especially using adult somatic cells. The objective of this study was to determine the developmental competence of NT embryos derived from various somatic cells embryonic stem (ES), amniotic epithelial, cumulus, or fetal fibroblast cells] and the nuclear transfer method, such as electro fusion or piezo microinjection, activation with chemical reagent [ionomycine/6-dimethylaminopurine (DMAP), calcium ionophore A23187/DMAP, or cycloheximide (CHX)] and reprogramming time (1, 2, or 4 h; in this study, the duration from injection or fusion to activation was defined as the reprogramming time). Our results showed that a 1-h reprogramming and activation with ionomycin/DMAP are suitable for NT in monkeys. Developing cleaved embryos up to the six-cell stage was similar among all experiments. However, beyond the eight-cell stage, developmental rates were higher in NT embryos reconstructed with fetal fibroblast cells and amniotic epithelial cells, and we were able to produce NT blastocysts from these cells. Interestingly, electro fusion is sufficient for amniotic epithelial cells and piezo microinjection is better suited for fetal fibroblast cells to produce NT blastocysts, thus suggesting that the best method for somatic cell NT may be different between cell types.

Collection and culture of primordial germ cells from cynomolgus monkeys (Macaca fascicularis).

Aim: To clarify the location of primordial germ cells (PGC) in an embryo of target-age and to examine the culture environment of the PCG. Methods: The days of ovulation and fertilization were estimated by measuring the serum concentration of estrogen. Pregnancy was confirmed by measurement of the serum concentration of the beta subunit of macaque chorionic gonadotropin and by ultrasonography. We also examined the location of PGC in the embryo at the time of retrieval. Results: Results showed that PGC in an embryo were in the hindguts at day 30 postfertilization, arrived at the genital ridges via mesenteries at approximately day 33 postfertilization, and colonized the gonads by day 36 postfertilization. Conclusions: In conclusion, embryos collected on day 33 postfertilization are more suitable for obtaining PGC from cynomolgus monkeys. The PGC collected from cynomolgus monkey fetuses were cultured under conditions for the derivation and culture of human embryonic germ cells; enzymatically dispersed single cells were cultured on a SIM thioguanine-resistant ouabain-resistant cells (STO) feeder layer with recombinant human leukemia inhibitory factor, recombinant human basic fibroblast growth factor and forskolin. The cells from genital ridges and mesenteries at day 33 postfertilization had alkaline phosphatase (ALP) activity in vitro for a maximum of 13 days. In contrast, ALP activity had been held for 2 months under the same culture condition when the cells were derived from the gonads at day 66 postfertilization. Derivation of an embryonic germ cell from a cynomolgus monkey was not achieved from these cultures. (Reprod Med Biol 2007; 6: 203-210).

Identification and characterization of stem cells in prepubertal spermatogenesis in mice.

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP(+) KIT(-) fraction of prospermatogonia has much higher repopulating activity than does the GFP(+)KIT(+) population in the adult environment. Interestingly, the GFP(+)KIT(+) population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte-Sertoli cell adhesion and migration of gonoyctes toward the basement membrane.